Treatment of cancer or tumors induced by the release of heat generated by various chains of magnetosomes extracted from magnetotactic bacteria and submitted to an alternative magnetic field

ABSTRACT

In this disclosure, we describe a method for the treatment of tumor(s) or tumor cell(s) or cancer(s) in a subject in need by the generation of heat. The latter is produced by chains of magnetosomes extracted from whole magnetotactic bacteria and subjected to an alternative magnetic field. These chains of magnetosomes yield efficient antitumoral activity whereas magnetosomes unbound from the chains or kept within the whole bacteria produce poor or no antitumoral activity. The introduction of various chemicals such as chelating agents and/or transition metals within the growth medium of the bacteria improves the heating properties of the chains of magnetosomes. Moreover, the insertion of the chains of magnetosomes within a lipid vesicle is also suggested in order to favor their rotation in vivo and hence to improve their heating capacity. The vesicle can contain an antitumoral agent together with the chains of magnetosomes. In this case, the agent is released within the tumors by heating the vesicle.

FIELD OF THE INVENTION

The present invention is related to the in vivo heat treatment of cellsor tissues, especially tumor(s) or tumor cells, using the heat generatedin situ by magnetic elements submitted to an alternative magnetic field.The invention is related in particular to heat therapy usinghyperthermia or thermoablation. The type of magnetic element describedin this disclosure is a chain of iron oxide nanoparticles synthesizedthrough a biological process.

BRIEF SUMMARY OF THE INVENTION

This invention describes a thermotherapy, which can be used to destroycancer, tumor(s), or tumor cells. The heat is generated by chains ofbacterial magnetosomes, which are extracted from magnetotactic bacteria.The chains of magnetosomes used in the thermotherapy may be obtained bycultivating the bacteria in the various following conditions:

(1) Magnetotactic bacteria (e.g. ATCC 700274) are cultivated in astandard growth medium (e.g. ATCC Medium 1653 or a growth medium similarto ATCC Medium 1653 suitable to grow the strain ATCC 700274).(2) Magnetotactic bacteria are cultivated in a growth medium, whichcontains the standard growth medium such as that mentioned in (1) andpreferably an additive, which is a transition metal. Examples oftransition metals, which can be used, are Cobalt, Nickel, Copper, Zinc,Manganese and Chrome.(3) Magnetotactic bacteria are cultivated in a growth medium, whichcontains the standard growth medium such as that mentioned in (1) andpreferably an additive, which is a chelating agent. By chelating agent,it is meant preferably an organic compound, which is a monodentate orpolydentate ligand able to form a complex with the cations derived fromiron or anyone of the other transition metals.(4) Magnetotactic bacteria are cultivated in a growth medium, whichcontains the standard ATCC growth medium such as that mentioned in (1)and the two additives mentioned in (2) and (3).

The presence of additives within the bacterial growth medium yieldsimproved magnetosome heating efficiency (both in solution and in vivo).Extracted chains of magnetosomes obtained by synthesizing the bacteriain either one of the four different growth media described in (1) to (4)can also be encapsulated within a lipid vesicle in the presence or notof an active principle and used as such in the thermotherapy.

BACKGROUND

Recently, great efforts have been devoted to synthesizing magneticnanoparticles, which are able to induce the production of heat when anoscillating magnetic field is applied to them (Duguet et al., Nanomed.,2006, 1, 157-168) and that can be easily manipulated using magneticfields. These features have led to the idea that magnetic nanoparticlesmay be helpful in the destruction or elimination of tumors throughhyperthermia or thermoablation or that they can be used to release drugsat specific localized regions of the body. This field of research isoften designated as alternative magnetic field (AMF) hyperthermia sinceit requires the application of an alternative magnetic field to inducethe production of heat by the nanoparticles. In previous work, the heathas been induced using chemically synthesized nanoparticles, mainly inthe form superparamagnetic iron oxide nanoparticles (SPION), which wereeither mixed in solution or mixed with cells or administered to a livingorganism. The anti-tumoral activity of these heated nanoparticles hasalso been evaluated both on animal models and clinically on humans. Anoverview of the work carried out previously is presented in thereferences listed hereafter (Bae et al., J. Controlled Release, 2007,122, 16-23; Ciofani et al., Med. Hypotheses, 2009, 73, 80-82; De Nardo,Clin. Cancer Res., 2005, 11, 7087s-7092s; De Nardo et al., J. Nucl.Med., 2007, 48, 437-444; Higler et al., Radiology, 2001, 218, 570-575;Ito et al., Cancer. Sci., 2003, 94, 308-313; Ito et al., J. Biosci.Bioeng., 2003, 96, 364-369; Ito et al, Cancer Lett., 2004, 212, 167-175;Ito et al., Cancer Immunol. Immun., 2006, 55, 320-328; Johannsen et al.,Int. J. Hyperthermia, 2005, 21, 637-647; Johannsen et al., Int. J.Hyperthermia, 2007, 52, 1653-1662; Jordan et al., Int. J. Hyperthermia,1993, 9, 51-68; Kawai et al., Prostate, 2005, 64, 373-381; Kawai et al.,Prostate, 2008, 68, 784-792; Kikumori et al., Breast Cancer Res. Treat.,2009, 113, 435-441, Maier-Hauff et al., J. Neurooncol., 2007, 81, 53-60;Oberdorster et al., Environ. Health Persp., 2005, 113, 823-839; Ponce etal., Int. J. Hyperthermia, 2006, 22, 205-213; Tai et al.,Nanotechnology, 2009, 20, 135101; Thisen et al., Int. J. Hyperthermia,2008, 24, 467-474).

At this time, there are at least three companies that develop cancertherapy using the heat generated by magnetic nanoparticles when thelatter are exposed to an alternative magnetic field. These companies areSirtex (an Australian company), Magforce (a German company) and AspenMedisys (an American company previously Aduro Biotech and TritonBiosystem). The patterns that have been published by these companiesdescribe various ways of using the heat generated by chemicallysynthesized magnetic nanoparticles for cancer therapy (Sirtex:US2006167313 or WO 2004/064921; Triton Biosystems now Aspen Medisys,LLC: US2003/0028071; Magforce: US2008/0268061).

Although significant progress has been made in the area of nanoparticlecancer therapy, concerns have been raised regarding the toxicity inducedby the presence of the chemically synthesized nanoparticles in the body(Habib et al., J. Appl. Phys., 2008, 103, 07A307-1-07A307-3). In orderto minimize the potential side effects arising during the clinicaltreatments, the quantity of nanoparticles administered needs to be assmall as possible while still retaining their desired effect. For that,magnetic nanoparticles have to generate a sufficiently large amount ofheat, i.e. significant specific absorption rates (SAR).

Therefore, there is a need for magnetic nanoparticles having a higherheating capacity than that usually obtained with chemically synthesizednanoparticles. This will be useful to reduce the amount of magneticmaterial needed to heat a biological tissue or cell. This can beachieved by using nanoparticles with either large volumes or with highmagnetocrystalline anisotropy (Hergt et al., J. Phys. Condens. Matter,2006, 18, 52919-52934).

There is also a need to develop magnetic nanoparticles that can havesuch good properties and the ability to target a tissue or a cell.

In part due to their large volume, the magnetosomes synthesized bymagnetotactic bacteria produce a larger amount of heat than thechemically synthesized nanoparticles when they are subjected to anoscillating magnetic field. This has been shown for bacterialmagnetosomes mixed in solution (Hergt et al., J. Phys. Condens. Matter,2006, 18, S2919-S2934; Hergt et al., J. Magn. Magn. Mater., 2005, 293,80-86; Timko et al., J. Mag. Mag. Mat., 2009, 321, 1521-1524). In theabove references, the type of bacterial magnetosomes used to carry outthe experiments has not been clearly identified.

The magnetosomes are intracellular, membrane-bounded, nanometer-sizedsingle-magnetic-domain crystals of the iron oxide magnetite (Fe₃O₄) oriron sulfide greigite (FeS₄) that are synthesized by magnetotacticbacteria. The magnetosomes composed of magnetite can become oxidized tomaghemite after extraction from the bacteria. The magnetosomes areusually arranged as a chain within the bacteria, but individualmagnetosomes can also be found. The bacteria appear to use themagnetosomes to navigate in the Earth's geomagnetic field and help themto locate and maintain optimal conditions for their growth and survival(Bazylinski et al., Nat. Rev. Microbiol., 2004, 2, 217-230).Magnetosomes and magnetosome magnetite crystals have been shown to beuseful in a number of scientific, commercial and health applications.For example, they can be used to detect single nucleotide polymorphism,to extract DNA or to detect magnetically bio-molecular interactions.They can also be used in immunoassay and receptor-binding assay or incell separation (Arakaki et al., J. R. Soc. Interface, 2005, 5,977-999). It has been suggested that bacterial magnetosomes could beinserted within liposomes for drug delivery purposes (Pattern numberU.S. Pat. No. 6,251,365B1). However, very few experimental proofs havebeen given in this pattern and the heating capability of such liposomehas not been demonstrated or suggested. The anti-cancerous activity of acomplex formed by bacterial magnetosomes and doxorubicin has been shownexperimentally (Sun et al., Cancer Lett, 2007, 258, 109-117). In thiscase, the anti-cancerous activity is due to the presence of doxorubicinand not to a treatment induced by heat. In the end, bacterialmagnetosomes have not been proven to be useful for in vitro or in vivoheat treatment of tumor or cancer cells.

Finally, two recent studies briefly address the issue raised by thepotential toxicity of bacterial magnetosomes in rats and don't reportany sign of toxicity (Sun et al., J. Nanosci. Nanotechnol., 2009, 9,1881-1885; Sun et al, Sun et al, Nanotoxicology, 2010, 4, 271-283).

DESCRIPTION OF THE INVENTION

Different types of bacterial magnetosomes (organized in chains or notand contained within the bacteria or extracted from the bacteria) can beefficient to generate heat in a solution when they are exposed to analternative magnetic field. However, as demonstrated in this disclosure,only magnetosomes organized in chains and isolated from magnetotacticbacteria yield efficient anti-tumoral activity. Indeed, bacterialmagnetosomes contained within whole AMB-1 magnetotactic bacteria andindividual magnetosomes (extracted from the bacteria and treated withsodium dodecyl sulfate (SDS) and heat) were also studied. Despite theirgood heating properties in solutions, these two types of bacterialmagnetosomes appeared to yield no or much less in vivo antitumoralactivity than the chains of magnetosomes according to the invention. Theimpact of the organization in chains of the bacterial magnetosomes onthe efficiency of the thermotherapy is an important contribution of theinvention.

The present invention is related to the in vivo treatment of tissues orcells, especially of tumor(s) or tumor cell(s), using the heat generatedin situ by chains of magnetosomes, which are isolated and extracted fromwhole magnetotactic bacteria. The type of tumor, which can be treated,is preferentially a solid tumor. These chains may be used as such orencapsulated within a vesicle. The heat is produced by submitting thechains of magnetosome to an alternative magnetic field (also calledoscillating magnetic field).

The present invention is also related to chains of magnetosomes for usein the treatment of tumor(s) by heat therapy, preferentially of a solidtumor. The chains may be used as such or encapsulated within a vesicle.

The present invention is also related to chains of magnetosomes as adrug, especially as a drug for anti-tumoral treatment. The chains may beused as such or encapsulated within a vesicle.

The invention is also related to the use of chains of magnetosomes as amean of heating, especially of a living tissue or living cells in vivo.

The invention is also related to the use of chains of magnetosomes as adrug which enables the treatment of tumor(s) and/or tumor cells througha heating method.

The following description of embodiments and features do apply to themethod of treatment and to the use of the chains of magnetosomes.

It is important to point out that the invention is related to theadministration of chains of magnetosomes to a patient in need. However,it is possible that after administration in the organism a small amountof chains of magnetosomes is altered; the alteration of these chains ofmagnetosomes may result in the formation of longer or shorter chainsthan those administered and less probably in the apparition ofindividual magnetosomes.

The magnetosomes administered during the therapy are in the form ofchains of magnetosomes. By definition, these chains of magnetosomes areisolated from the magnetotactic bacteria. This means that they are notcontained within the bacteria. Preferably, the chains have beenextracted from the bacteria used for their production and isolated fromthe cellular fragments. These chains of magnetosomes contain preferablybetween 2 and 30 magnetosomes, typically between 4 and 20 magnetosomes.Most of the magnetosomes belonging to these chains possesscrystallographic directions and preferably also easy axes orientated inthe direction of the chain elongation, which is usually [111](Alphandéry et al., ACS Nano., 2009, 3, 1539-1547). Consequently, thechains of magnetosomes possess a magnetic anisotropy, which is strongerthan that of individual magnetosomes. As a result, strong aggregation ofthe chains of magnetosomes is prevented. When several chains ofmagnetosomes containing typically between 4 and 20 magnetosomesinteract, it results in the formation of a longer chain of magnetosomes,containing typically more than 4 to 20 magnetosomes. The length of achain of magnetosomes is preferably less than 1200 nm, more preferablyless than 600 nm, most preferably less than 300 nm. The arrangement inchains of the magnetosomes yields several properties, which areadvantageous for in vivo heating. Due to their arrangement in chains,the magnetosomes are not prone to aggregation and also possess a stablemagnetic moment. Both of these properties favor the rotation of thechains of magnetosomes and therefore the production of heat through thismechanism. The arrangement in chains of the magnetosomes also providesan interaction with the eukaryotic cells, which is advantageous due totheir low level of aggregation. This interaction results in aninternalization of the chains of magnetosomes within the eukaryoticcells. For example, as described in more details in example 4, asignificant percentage of cells become magnetic when the chains ofmagnetosomes are mixed with the cells while the alternative magneticfield is applied. In an embodiment, the chains of magnetosomes penetratewithin the eukaryotic cells when the alternative magnetic field isapplied, thus enabling the destruction of the cells through themechanism of intra-cellular hyperthermia. This mechanism is potentiallymore efficient than extra-cellular hyperthermia since it destroys thecells from the inside. On the other hand, a very small percentage ofmagnetic cells is obtained when the cells are mixed in the presence ofthe individual magnetosomes while the alternative magnetic field isapplied, suggesting that the individual magnetosomes remain outside ofthe eukaryotic cells, yielding a less efficient mechanism of celldestruction.

Magnetosomes are defined as magnetic iron oxide nanoparticles made ofmagnetite, maghemite or of a composition which is intermediate betweenmaghemite and magnetite. The magnetosomes are also characterized by thepresence of a biological membrane, which surrounds them. The presence ofamino groups at the surface of the magnetosome membrane enables acoupling with various bioactive macromolecules and yieldsbiocompatibility (Xiang et al., Lett. Appl. Microbiol., 2007, 6, 75-81;Sun et al., Cancer Lett., 2007, 258, 109-117; Sun et al., Biotech.Bioeng., 2008, 101, 1313-1320).

In an embodiment, the magnetosomes belonging to the chain are surroundedby a biological membrane. The magnetosomes may be bound to each othervia a biological filament whose structure is only partly known accordingto A. Komeili, Ann. Rev. Biochem. 2007, 76, 351-366.

In one embodiment the magnetosomes are synthesized biologically bymagnetotactic bacteria such as Magnetospirillum magneticum strain AMB-1,magnetotactic coccus strain MC-1, three facultative anaerobic vibriosstrains MV-1, MV-2 and MV-4, the Magnetospirillum magnetotacticum strainMS-1, the Magnetospirillum gryphiswaldense strain MSR-1, a facultativeanerobic magnetotactic spirillum, Magnetospirillum magneticum strainMGT-1, and an obligate anaerobe, Desulfovibrio magneticus RS-1.

The sizes or mean sizes of the individual magnetosomes contained withinthe chains of magnetosomes may vary depending in particular on thestrain of bacteria, the bacterial growth medium and/or the bacterialgrowth conditions. Most frequently, the magnetosomes are monodomainnanoparticles (i.e. they possess only one magnetic domain) with sizeslying between about 10 nm and about 120 nm, preferably between 10 nm and70 nm, most probably between 30 nm and 50 nm. The magnetosome sizedistribution can vary quite significantly depending on the bacterialstrain and bacterial growth conditions. In the AMB-1 species, themajority of the magnetosomes possess sizes lying between 30 nm and 50nm. Increase in sizes may be obtained when the production of themagnetosomes is carried out in the presence of one or several of theadditives such as those described in the invention. The large sizes ofthe magnetosomes result in ferrimagnetic behaviors at the temperaturesreached during the treatment. It also yields a thermally stable magneticmoment. Hence, the movement of the magnetosomes in the organism couldpotentially be controlled by applying an external magnetic field. Due totheir stable magnetic moment, the magnetosomes should produce a bettermagnetic response than the smaller superparamagnetic iron oxidenanoparticles (SPION) currently used for the medical applications, whichpossess a thermally unstable magnetic moment. The magnetosomes, whichare large monodomain nanoparticles, also possess better heatingproperties than most chemically synthesized nanoparticles (usually inthe form of SPION) when they are suspended in a solution and exposed toan alternative magnetic field.

In one embodiment, the magnetosomes possess a narrow size distributionwhen the magnetotactic bacteria are grown under optimum conditions.

In one embodiment, a step of size selection can be carried out usingeither a magnetic field of various intensities (0.05-1 T), a sizeselection chromatography technique (using for example a column of thetype Sephacryl S1000) or a centrifugation technique, which enables toget rid of the smallest magnetosomes remaining in the supernate. Usingmagnetosomes with sizes lying in a given range can also be helpful inorder to introduce them in vesicles of a given size for example.

In a specific embodiment, the method of the present invention useschains of magnetosomes encapsulated within a vesicle, especially a lipidvesicle. The encapsulation of the chains of magnetosomes yields improvedheating properties and also reduces the risks of toxicity by preventinga direct contact between the chains of magnetosomes and the organism.The rotation of the chains of magnetosomes in vivo may be improved bytheir encapsulation within a lipid vesicle or a similar type ofstructure.

In one embodiment, the lipid vesicle is a small unilamellar vesicle(SUV, diameter<100 nm), containing a reduced amount of small chains ofmagnetosomes. Compared with larger vesicle, the SUVs possess severaladvantages. For example, they are less recognizable by the macrophages(Genc et al., Langmuir, 2009, 25, 12604-12613).

In another embodiment, the lipid vesicle is a large unilamellar vesicle(LUV, diameter lying between 100 nm and 1 μm) or a giant unilamellarvesicle (GUV, diameter >1 μm). It is more preferably a LUV forintravenous injection. In the cases of LUV or GUV, the capacity ofmagnetosome uptake is significantly larger than that of the SUV andhence the heating efficiency is larger.

In one embodiment, the lipid vesicle is a liposome, which ismultilamellar.

In one embodiment, the lipid vesicle is composed of a single lipid witha neutral charge such as DOPC(1-Oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine), DMPC(dimyristoylphosphatidylcholine), DPPC (Dipalmitoylphosphatidylcholine),DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DMPE (dimyristoylphosphatidylethanolamine or DPPE (dipalmitoylphosphatidylethanolamine).

In another embodiment, the lipid vesicle is composed of neutral lipidssuch as those mentioned above mixed with charged lipids such as DOPG(1,2-Dioleoyl-sn-glycero-3-[Phospho-rac-(1-glycerol)]), DPPG(Dipalmitoylphosphatidylglycerol). Lipids with various charges are mixedtogether in order to optimize the surface charge of the vesicle. Indeed,the latter is an important parameter for the encapsulation of the chainsof magnetosomes with the vesicles and for the internalization of thevesicles within the cells (Martina et al., J. Am. Chem. Soc., 2005, 127,10676; Tai et al., Nanotechnology, 2009, 20, 13501).

The method aims at providing in vivo heat therapy, includinghyperthermia and thermoablation.

In one embodiment the method described in the present invention shows away to partially or totally destroy the tumor cells or the tumor by anincrease of the temperature in the tumor of less than ˜10′C above thephysiological temperature (37° C.), a technique usually calledhyperthermia. In a preferred embodiment, the temperature in the tumorreached during hyperthermia lies between about 37° C. and about 45° C.,preferably between about 40° C. and about 45° C., more preferably atabout 43° C.

In another embodiment the method of the present invention shows a way todestroy tumor cells or tumor by an increase of the temperature in thetumor of more than 10° C. above physiological temperature (37° C.), atechnique usually called thermoablation. The temperature reached duringthermoablation lies between about 45° C. and about 100° C., morepreferably between about 45° C. and about 70° C.

In a preferred embodiment, the temperature in the tumor reached duringthermoablation lies between about 45° C. and about 55° C., preferablybetween about 50° C. and about 55° C., most preferably at about 53° C.,54° C. or 55° C.

Since the heat is produced very locally (at the nanometer scale)relatively high temperatures could be reached locally during thetreatment.

The temperatures indicated above are temperatures reached within thetumor(s), the tumor tissue and/or their environment. The temperaturewithin the tumor cells (i.e. near the internalized magnetosomes) couldbe higher.

An object of the present invention is a method for the partial or totaldestruction of tumor cells or of a tumor. The tumor cells can be killedor lose their ability to multiply indefinitely when the heat treatmentdescribed in this invention is applied to them. Since tumor cells aremore susceptible to heat than healthy cells (See for example: Overgaardet al., Cancer, 1977, 39, 2637-2646), the thermotherapy described inthis disclosure could selectively destroy tumor cells.

The method of the present invention describes a heat treatment whichinduces the partial or total destruction of the tumor cells and/or ofthe tumor(s). The heat treatment is generated by application of analternative magnetic field. This magnetic field induces the productionof heat by the chains of magnetosomes (encapsulated or not in avesicle).

In one embodiment, the alternative magnetic field applied during thetreatment is characterized by a frequency lying between about 50 kHz andabout 1000 kHz, preferably between about 100 kHz and about 500 kHz, morepreferably between about 100 kHz and about 200 kHz.

In another embodiment, the magnetic field is characterized by a strengthlying between about 0.1 mT and about 200 mT, preferably between about 1mT and about 100 mT, more preferably between about 10 mT and about 60mT, typically between about 10 mT and about 50 mT.

The maximum value of the magnetic field strength is determined by thevalue at which it becomes toxic for the organism (i.e. essentially whenit generates Foucault's currents). It may be possible that magneticfields of strengths higher than 200 mT can be used in the therapy ifthey are shown to be non toxic.

In another embodiment, the method of the present invention ischaracterized by the length of time during which the magnetic field isapplied. This length of time may be between about 1 second and about 6hours, preferably between about 1 minute and about 1 hour, preferablybetween 0.5 and 30 minutes, most preferably between 1 minute and 30minutes.

The heat treatment is preferably applied to anesthetized patients.Therefore, the time during which the treatment is carried out may beequal or less than the length of time of the anesthesia. A heattreatment can thus potentially be carried out during more than 6 hours,for example if a patient is anesthetized during more than 6 hours.

In another embodiment, the method of the present invention ischaracterized by the quantity of magnetosomes used during the therapy.This quantity of magnetosomes is related to the quantity of iron oxidecontained in the suspension of chains of magnetosomes. This quantity isestimated by measuring the amount of iron oxide present in thesuspension of chains of magnetosomes, which is injected. It lies betweenabout 0.001 mg and about 100 mg of iron oxide, preferably between about0.01 mg and about 100 mg of iron oxide, more preferably between about0.01 mg and about 10 mg of iron oxide, more preferably between 0.1 and10 mg of iron oxide, typically between 0.1 and 1 mg of iron oxide. Thequantity of magnetosomes, which needs to be injected, essentiallydepends on the volume of the treated tumor, the temperature requiredduring the treatment and the method of injection. The largest tumorvolume and the highest tumor temperature require the largest quantity ofmagnetosomes administered. Moreover, if the magnetosomes areadministered intravenously (or otherwise from the outside of the tumorlocation(s)), more chains of magnetosomes might be needed than if theyare directly administered within or close to the tumor(s).

In another embodiment, the administration of the chains of magnetosomescan be carried out at different speed depending on the targeted tumor(s)and on the concentration of the suspension of chains of magnetosomesadministered. For example, administration of the suspension of chains ofmagnetosomes directly within brain tumor(s) might require a slower speedof injection than intravenous injection or than an injection within atumor localized at the skin surface. The injection of a moreconcentrated suspension of chains of magnetosomes might require a slowerspeed of injection than that of a less concentrated suspension of chainof magnetosomes.

The speed of injection preferably lies between 0.1 μl/min and 1liter/min, more preferably between 1 μl/min and 100 ml/min, mostpreferably between 1 μl/min and 10 ml/min, where the indicated volume isthe volume of the suspension of chains of magnetosomes administered.

In another embodiment, the concentration of the suspension of chains ofmagnetosomes typically lies between 1 μg/ml and 100 mg/ml, preferablybetween 10 μg/ml and 50 mg/ml, where this concentration represents thequantity of iron oxide (preferentially maghemite) contained within thesuspension. In another embodiment, the chains of magnetosomes are mixedwith a solvent, which stabilizes the chains of magnetosomes. The pH ofthe suspension can be adjusted and/or cations and/or anions can be addedto the suspension containing the chains of magnetosomes to stabilizethis suspension.

In another embodiment, the administration of the chains of magnetosomesto the patient is repeated. The number of repetition depends on thequantity of magnetosomes, which is administered at once. If only a smallquantity of chains of magnetosomes is administered at once, theadministration step might be repeated several times until the desiredamount of magnetosomes is administered to a patient.

In another embodiment the heat treatment started by application of thealternative magnetic field is repeated. The successive heat treatmentsapplied after administration of a given amount of chains of magnetosomeare called a heat cycle. The given amount of magnetosomes used for eachheat cycle may have been administered through a single administration orthrough several successive administrations as explained above. Thedifferent heat treatments within a heat cycle are separated one fromanother by a resting time. The resting time may be equal to 1 second orlonger than 1 second, preferably equal to 1 minute or longer than 1minute, more preferably equal to 10 minutes or longer than 10 minutes,preferably equal to or longer than 30 minutes.

In an embodiment, the different heat treatments within a heat cycle areseparated one from another by a longer resting time than that mentionedabove. This resting time may lie between 1 day and 15 days.

In an embodiment, the heat cycle is repeated 1 to 648000 times, inparticular 1 to 1000 times, more particularly 1 to 100 times, typically1 to 10 times. The highest repetition rate of 648000 times is estimatedby assuming that the treatment is carried out for a very short time,typically about one second, during 15 days with a very short restingtime, typically about one second resting time separating each treatment.The number of repetition of the treatment depends on the length of timeof the treatment. Preferentially the longer the treatment is the lessrepetition is needed provided the other parameters of the therapy (suchas the strength and or frequency of the applied magnetic field) arefixed.

According to the invention, a cession includes the sequences ofadministering a given amount of chains of magnetosomes to a patient andthe generation of heat through application of the magnetic field (aswell as other optional sequences as described hereafter). Differentcessions may be carried out on the same patient. These cessions may beseparated one from another by a length of time, which is sufficientlylong. This length of time may be equal to 1 day or longer than 1 day,preferably equal to 15 days or longer than 15 days, more preferablyequal to 1 month or longer than 1 month.

In order to optimize the efficiency of the thermotherapy, one needs toadjust the following parameters, i.e. the amount of chains ofmagnetosomes used during the therapy, the frequency and/or strength ofthe applied magnetic field, the length of time of the treatment, thenumber of times that the treatment is repeated during one “cession” andthe number of “cessions”. These parameters may depend on specificproperties of the tumor which is targeted, i.e. for example on its size,its resistance to the thermotherapy and its viscosity. For a tumor witha large volume and/or high resistance in temperature and/or highviscosity, one might consider to increase the amount of magnetosomesinjected and/or the strength/frequency of the applied magnetic fieldand/or the number of repetitions of the treatment. In this case, onemight also consider encapsulating the bacterial magnetosomes within avesicle to favor the production of heat. In an embodiment, theparameters of the thermotherapy are adjusted to optimize the efficiencyof the treatment of the tumor to be treated.

In still another embodiment, the values of these parameters also dependon the number of tumors and the presence of metastases, which need to betreated. For a patient in a state of advanced cancer, i.e. withmetastasis and/or an important number of tumors, the amount of chains ofmagnetosomes needed will be higher than for a single tumor. Instead ofincreasing the quantity of magnetosomes administered, one might alsoconsider to increase the length of time of the treatment, the strengthof the magnetic field applied during the treatment (to reach highertemperatures) or the number of times that the treatment is repeated.

The method aims at treating cancers, more preferably solid tumors.Examples of cancers, which can be treated with this type ofthermotherapy, include prostate cancer (Kawai et al., Prostate, 2008,68, 784-792), esophageal cancer, pancreatic cancer, breast cancer(Kikumori et al., Breast Cancer Res. Treat., 2009, 113, 435-441), braincancer (Thiesen et al., Int. J. Hyperthermia, 2008, 24, 467-474) andskin cancer (Ito et al., Cancer Sci., 2003, 94, 308-313).

Another object of the invention is a method for the production of chainsof magnetosomes wherein magnetotactic bacteria are cultivated in agrowth medium containing at least an iron source, such as an ironquinate solution, and additives such as other transition metals thaniron and/or chelating agents as defined therein. As an example, thegrowth medium contains the ingredients mentioned in example 1. Theseadditives produce in specific conditions an increase in the sizes of themagnetosomes and/or in the length of the chains of magnetosomes. Theyconsequently enhance the heating capacity of the chains of magnetosomeswhen they are exposed to an alternative magnetic field.

In one embodiment, the magnetotactic bacteria are cultivated in a growthmedium containing the standard growth medium of magnetotactic bacteria,such as that described in example 1 for the AMB-1 species, and anadditive, which is a transition metal, such as for example Cobalt,Nickel, Copper, Zinc, Manganese, Chrome or a mixture of two or more ofthese metals.

In an embodiment, the doping of the magnetosomes with a transitionmetal, e.g. cobalt, is carried out by adding about 0.02 μM to 1 mM,preferably 0.02 μM to 200 μM, preferably 1 μM to 100 μM, preferably 2 to20 μM solution of transition metal (e.g. cobalt) within the growthmedium of the magnetotactic bacteria. Such solution could be for examplea solution of quinate cobalt added to the standard growth medium of themagnetotactic bacteria, e.g. of the AMB-1 species (ATCC 70027),following the same method as that used by Staniland et al (S. Stanilandet al., Nature Nanotech. 2008, 3, 158-162). The magnetotactic bacteriasynthesized in the presence of cobalt, e.g. cobalt quinate, or anothertransition metal possess improved magnetic properties even when thepercentage of cobalt doping is lower than 2% (S. Staniland et al.,Nature Nanotech., 2008, 3, 158-162). In the above study by Staniland etal., the change of the magnetic properties in the presence of cobalt wasobserved for whole magnetotactic bacteria and not for the chains ofmagnetosomes extracted from the magnetotactic bacteria. The improvementof the magnetic properties of Co-doped magnetosomes arranged in chainsand extracted from magnetotactic bacteria, yielding improved heatingcapability, is a contribution of this invention. For chemicallysynthesized nanoparticles, a percentage of Co-doping larger than about10% is usually necessary to observe large changes in the magneticproperties (A. Franco et al., J. Mag. Mag. Mat., 2008, 320, 709-713; R.Tackett et al., J. Mag. Mag Mat., 2008, 320, 2755-2759). This indicatesthat Co-doped magnetosomes could possess improved heating capacitycompared with undoped magnetosomes even for a low percentage of cobaltdoping.

In another embodiment, the magnetotactic bacteria are cultivated in thepresence of a chelating agent. Without being fully explained by theory,it is thought that the chelating agent binds the cations derived fromiron or any one of the other transition metals used as additives, andconsequently improves the penetration of iron and/or of anothertransition metal within the magnetotactic bacteria. This process yieldsmagnetosomes with improved heating properties.

In an embodiment, a suspension containing about 0.02 μM to 1 mM,preferably 0.02 μM to 400 μM, preferably 0.02 to 200 μM, preferably 1 μMto 100 μM, most preferably 2 to 20 μM of an iron chelating agent isadded to the growth medium.

In an embodiment, the chelating agent is a molecule, which contains oneor several carboxylic acid functional groups such as ALA (alpha lipoicacid), calcein, carboxyfluoresceine, deferasirox, dipicolinic acid, DTPA(Diethylene triamine pentaacetic acid), EDTA (ethylene diamine tetraacetic acid), folic acid or vitamin B9, lactic acid, rhodamine B,carboxymethyl-dextran (polymers), dipicolinic acid or oxalic acid,citric acid or citrate functional groups, such as BAPTA(Aminophenoxyethane-tetraacetic acid), CDTA(cyclohexane-1,2-diaminetetra-acetic acid), EDDHMA (ethylene diaminedi-(o-hydroxy-p-methylphenyl) acetic acid), CaNa₂-EDTA, EDTCA (ethylenediamine tetra-acetic acid plus Cetavlon, an ammonium surfactant), EDDA(ethylene diamine-N,N′-diacetic acid), EDDHA(ethylenediamine-N,N′-bis(2-hydroxyphenylacetic acid), EGTA (ethyleneglycol-bis-(β-amino-ethyl ether) N,N, N′, N′-tetra-acetic acid), HEDTA(N-(2-hydroxyethyl)-ethylenediaminetriacetic acid), HEEDTA(Hydroxy-2-ethylenediaminetriacetic Acid), NTA (nitrilotriacetate) orphenolic acid.

In another embodiment, the chelating agent is a molecule, which containsone or several alcohol functional groups, such as catechol or theirderivatives or one or several amino-alcohol functional groups, such asdopamine, deferiprone, deferoxamine, desferrioxamine, or one or severalamino-carboxylic acid or ketone functional groups, such as doxorubicine,caffeine, D-penicillamine, pyrroloquinoline, HEIDA(hydroxyethylimino-N,N-diethanoic acid).

In one embodiment, the chelating agent is a molecule, which contains aphosphonate or a phosphonic acid functional group, such as AEPN(2-Aminoethylphosphonic acid), AMP (Amino-tris-(methylene-phosphonicacid)), ATMP (Amino tris(methylene phosphonic acid)), CEPA(2-carboxyethyl phosphonic acid), DMMP (Dimethyl methylphosphonate),DTPMP (Diethylenetriamine penta(methylene phosphonic acid)), EDTMP(Ethylenediamine tetra(methylene phosphonic acid)), HEDP (1-HydroxyEthylidene-1,1-Diphosphonic Acid), HDTMP (Hexamethylenediamine tetra(methylene phosphonic acid)), HPAA (2-Hydroxyphosphonocarboxylic acid),PBTC (Phosphonobutane-tricarboxylic acid), PMIDA(N-(phosphonomethyl)iminodiacetic acid), TDTMP (Tetramethylenediaminetetra(methylene phosphonic acid)), ADP (adenosinediphosphoric acid) or1-{12-[4-(dipyrrometheneborondifluoride)butanoyl]amino}dodecanoyl-2-hydroxy-sn-glycero-3-phosphate, asodium salt L-α-phosphatidic acid, a sodium salt1-palmitoyl-2-(dipyrrometheneborondifluoride)undecanoyl-sn-glycero-3-phospho-L-serine).

In another embodiment, the chelating agent is a molecule, which containsa bis, tris or tetra-phosphonate, or a bis, tris or tetra-phosphonicacid functional group, such as a 1-hydroxymethylene-bis-phosphonic acid,propane triphosphonic acid, (nitilotris(methylene))trisphosphonic acid,(phosphinylidynetris(methylene)) trisphosphonic acid. Examples of1-hydroxymethylene-bis-phosphonic acids include alendronic acid(Fosamax®), pamidronic acid, zoledronic acid, risedronic acid,neridronic acid, ibandronic acid (Bondronat®), minodronic acid and othercompounds described in the literature (L. Wilder et al, J. Med. Chem.,2002, 45, 3721-3728; M. Neves, N. Med. Biol., 2002, 29, 329-338; H.Shinoda et al, Calcif. Tissue Int., 1983, 35, 87-89; M. A. Merrel, Eur.J. Pharmacol., 2007, 570, 27-37). For 0.4 μM or 4 μM neridronic acid,alendronic acid and residronic acid introduced in the bacterial growthmedium, it has been observed herein that the percentage of magnetosomeslarger than 45 nm becomes larger than for the magnetosomes synthesizedin the absence of bisphosphonic acid. Chains of magnetosomes synthesizedin these conditions consequently possess improved heating properties.

In another embodiment, the chelating agent is a molecule, which containsa sulfonate or sulfonic acid functional group or BAL (Dimercaprol) suchas BPDS (bathophenanthrolinedisulfonate or4,7-di(4-phenylsulfonate)-1,10-phenanthroline), DMPS(Dimercapto-propoane sulfonate or 2,3-dimercapto-1-propanesulfonicacid), sulforhodamine 101, DMSA (Dimercatptosuccinic acid).

Other examples of chelating agents are polydentate ligands for examplehemoglobin, chlorophyll, porphyrin and organic compounds containingpyrrolic rings.

In another embodiment, the magnetotactic bacteria are cultivated in agrowth medium, which contains both a chelating agent and a transitionmetal.

In a preferred embodiment, cobalt is used as the transition metal,preferably used in combination with a chelating agent selected from abisphosphonic acid (neridronic acid, alendronic acid or risedronicacid), rhodamine or EDTA.

The method of treatment of the present invention comprises the followingsteps of:

-   -   (i) providing to a mammal chains of magnetosomes;    -   (ii) optionally, targeting the chains of magnetosomes in the        tissue, the tumor(s) and/or tumor cells to be treated;    -   (iii) optionally, detecting the chains of magnetosomes in the        tissue, tumor(s) or tumor cells to be treated;    -   (iv) heating by application of an alternative magnetic field;    -   (v) optionally, removing the chains of magnetosomes from the        tissue, tumor(s), tumor cells and/or the body.

In all the following embodiment and preferred embodiment, the chains ofmagnetosomes can be encapsulated in a vesicle or not.

Steps (iii) and (iv) could also be carried out in any order, forexample:

-   -   Step (iii) then step (iv); or    -   Step (iv) then step (iii)

Steps (ii), (iii), and (iv) could be carried out simultaneously orconsecutively.

Steps (ii) and (iv) could be carried simultaneously.

Steps (iii) and (iv) could be carried out simultaneously.

By mammal it is intended to mean any mammal also including human.

In an embodiment, the chains of magnetosomes of step (i) could be chainsof magnetosomes existing in the body, for example those remaining aftera first cycle of treatment.

In another embodiment, step (i) could be preceded by a step (i′) ofadministering the chains of magnetosomes to a mammal.

In an embodiment, step (i′) is carried out in such a way that the chainsof magnetosomes are administered far from the cells or tissue to betreated. For example, they are injected intravenously in the blood oradministered in another organ than that containing the tumor.

In an embodiment, step (i′) is carried out in such a way that the chainsof magnetosomes are administered close to the cells or tissue to betreated.

In still another embodiment, step (i′) is carried out in such a way thatthe chains of magnetosomes are administered within the cells and/ortumor(s) to be treated.

The distance between the zone of administration of the suspension ofchains of magnetosomes and the tumor location can vary depending onwhether or not it is possible to inject directly the magnetosomes withinthe tumor(s). For example, the tumor(s) could be located too close to avital organ. In this case, a direct injection of the chains ofmagnetosomes within the tumor(s) would not be possible.

The method of the present invention may also contain a second step oftargeting the tumor cells or tumor(s) to be treated. This step isparticularly important if the administration of the chains ofmagnetosomes (encapsulated in a vesicle or not) is not directly carriedout within the tumor. This is often the case when the type of injectionchosen is intravenous. The aim of the targeting step is to position thechains of magnetosomes within the environment of the tumor cells and/orof the tumor(s) and/or within the tumor cells and/or tumor(s).

In one embodiment, the step of targeting is carried out by using amagnetic field, which guides the chains of magnetosomes (encapsulated ornot in a vesicle) in the environment of the tumor cells and/or of thetumor(s) and/or in the tumor cells and/or tumor(s). This type oftargeting is designated as magnetic targeting.

In still another embodiment, two different approaches may be followed toguide the chains of magnetosomes magnetically within the environment ofthe tumor(s) and/or within the tumor(s) itself. On the one hand, amagnetic field is applied outside of the body of a patient and itsorientation is adjusted to enable the chains of magnetosomes to followthe right path until they reach the tumor location. This type ofmagnetic targeting is designated as an “active” targeting since it maynecessitate to change and adjust the characteristics of the magneticfield applied during the targeting step. An adapted MRI instrument inwhich either the patient or the magnetic field could be orientated inany direction might be used for this targeting step. On the other hand,a magnet could be positioned within or near the tumor location in orderto attract the chains of magnetosomes within the tumor and/or theenvironment of the tumor. In this case, magnetic targeting wouldessentially be passive (i.e. one would essentially wait for theaccumulation of the chains of magnetosomes within the tumor(s) and/ortumor environment).

In another embodiment, the step of targeting the tumor(s) is realized byattaching a biological and/or chemical targeting molecule, which targetsthe tumor(s), to the chains of magnetosomes or to the vesicle containingthe chains of magnetosomes. This targeting molecule is such that itspecifically recognizes the tumor cells. This type of targeting isdesignated as molecular targeting.

In an embodiment, this targeting molecule is an antibody, whichspecifically recognizes the tumoral cells.

In another embodiment, PEG or folic acid is used as the targetingmolecule.

In an embodiment, the coating of the surface of the chains ofmagnetosomes or that of the vesicle containing the chains ofmagnetosomes is realized by using poly (ethylene glycol) PEG, and/orfolic acid, and/or an antibody. The presence of these molecules may notonly enable to target specific cells but also favor intracellular uptakeand/or enable to avoid the recognition of the chains of magnetosomes bymacrophages (Allen et al, Trends in Pharmacological Sciences 1994, 15,215-220; Blume et al., Biochim. Biophys. Acta 1990, 1029, 91-97; Gabizonet al., Biochim. et Biophys. Acta, 1992, 1103, 94-100; Zhang et al.,Biomaterials 2002, 23, 1553-1561).

The step of targeting the tumor(s) could be realized by using acombination of both of the techniques mentioned above

-   -   magnetic targeting;    -   molecular targeting; or    -   magnetic targeting and molecular targeting.

In an embodiment, the method of detection of the bacterial magnetosomeswithin the organism (i.e. within the tumor or elsewhere) uses either MRIor another technique such as fluorescence. Such technique is either usedto verify that the chains of magnetosomes (encapsulated in a vesicle ornot) have reached the site of interest before the treatment induced byheat is started and/or to verify that the chains of magnetosomes are ontheir way to target the tumor and/or to verify that they are eliminatedproperly and/or to verify that they have been administered successfully.

In a very interesting embodiment, the magnetic field and especially themagnetic field used for the heating is used to internalize or improvethe internalization of the chains of magnetosomes within the tumorcells.

It is also possible, when the chains of magnetosomes are in the tumor orin the tumor cells, to use the magnetic field as a “final approach” toadjust the position of the chains of magnetosomes (encapsulated in avesicle or not) within the tumor in order to reach maximum heatingefficiency and/or anti-tumoral activity during the treatment induced byheat.

In still another embodiment, the chains of magnetosomes (encapsulated ina vesicle or not) are detected by MRI. The detection of wholemagnetotactic bacteria by MRI has been demonstrated (R. Benoit et al.,Clin. Cancer. Res. 2009, 15, 5170-5177). This embodiment concerns thedetection of the chains of magnetosomes.

In an embodiment, the chains of magnetosomes are used as contrastsagents, which can easily be detected by MRI due to their specificproperties (composition in iron oxide and/or good crystallinity).

In still another embodiment, the chains of magnetosomes are detectedusing a fluorescence detection technique. In this case, the chains ofmagnetosomes are modified by the presence of a fluorescent molecule orfluorescent tag, which is positioned at the surface and/or near thesurface and/or within one or several of the bacterial magnetosomesbelonging to the chains of magnetosomes. Such fluorescent molecule couldbe rhodamine, calceine, fluorescein, ethidium bromide, green or yellowfluorescent protein, coumarin, cyanine or the derivatives of theselisted molecules.

In another embodiment, the fluorescent molecules are positioned withinthe magnetosomes, at their surface or near their surface by cultivatingthe magnetotactic bacteria in the presence of these fluorescentmolecules. For example, fluorescent magnetosomes are obtained bycultivating the magnetotactic bacteria in the presence of about 0.1 μMto about 1 mM solution of fluorescent molecules. Fluorescentmagnetosomes can be obtained, for example by cultivating themagnetotactic bacteria in the presence of about 40 μM to about 400 μMsolution of Rhodamine.

In another embodiment, the fluorescent molecules are bound to the chainsof magnetosomes. This may be done by chemically attaching thefluorescent molecules to the surface of the magnetosomes.

In still another embodiment, the magnetosomes have fluorescent moleculesbound at their surface and contained inside them. This may be obtainedby using both of the techniques mentioned above to produce thefluorescent magnetosomes.

In still another embodiment, the vesicle containing the bacterialmagnetosomes is made fluorescent by attaching a fluorescent molecule atthe surface of the vesicle.

In an embodiment, the fluorescence of the chains of magnetosomes isexcited and detected using an excitation/detection scheme, which is suchthat it positioned outside of the organism. This type ofexcitation/detection scheme would be used if the tumor is located nearthe skin surface. For example, an optical fiber could be placed justabove the tumor near the skin surface to excite the modifiedmagnetosomes and collect the light emitted by them.

In another embodiment, the excitation and/or detection of the modifiedmagnetosomes is carried out by inserting within the organism a piece ofequipment (such as an optical fiber), which reaches the tumor and/or thetumor environment and is able to excite and/or detect the fluorescenceof the modified magnetosomes.

In an embodiment, step (v) is a step of removal of the magnetosomes fromthe tissue, the tumor(s), the tumor cells and/or the body. A technique,which guides the chains of magnetosomes outside of the organism, isused. The bacterial magnetosomes are either directly removed from thetumor(s) (for example chirurgically by making a hole, which provides apath for the bacterial magnetosomes to leave the tumor location andreach the outside of the organism). The bacterial magnetosomes couldalso be removed from the tumor location and driven towards other organssuch as the liver to be eliminated from the body.

In still another embodiment, the technique described in the aboveembodiment uses a magnetic field which drives the magnetosomes outsideof the tumor(s) and of the body.

In still another embodiment, the removal of the chains of magnetosomesfrom the tumor location is carried out by adjusting the charge surfaceof the chains of magnetosomes.

In still another embodiment, the chains of magnetosomes are negativelycharged.

In an embodiment, the fluorophore is also a chelating agent and is usedas an additive during the growth of the magnetotactic bacteria andmagnetosome production. In a preferred embodiment, rhodamine is used aschelating agent and fluorophore.

The therapeutic method then contains the heat treatment per se asdescribed above. This method thus enables a localized treatment oftumors and/or tumor cells and minimizes the destruction of healthycells. Therefore, it provides an improvement compared with chemotherapyor other techniques of cancer treatments, which do not usuallyspecifically target and destroy tumor cells.

In another embodiment, the heat treatment is combined with chemotherapy.

Such a combination of two treatments may be carried out by encapsulatingthe chains of magnetosomes within a vesicle, preferably a lipid vesicle,in the presence of an active principle, which is an anti-tumoral oranti-cancerous substance. In this case, the lipids forming the vesiclesare characterized by a phase transition temperature (the temperature atwhich the lipids forming the vesicle lose their bi-layer organization)lying between 20° C. and 60° C. The active principle is released in thetumor cells or in the tumors or in the environment of the tumor cells orin the environment of the tumors by heating the chains of magnetosomesand hence the vesicle under the application of an alternative magneticfield.

The vesicles, especially lipid vesicles containing the chains ofmagnetosomes according to the invention and possibly an active principleaccording to the invention are also an object of the present invention.

It is also possible to carry out the method of the invention incombination with x-ray and/or radio-therapy and/or chemotherapy and/or achirurgical operation and/or another type of cancer treatment.

In an embodiment, a chirurgical operation is carried out to partly ortotally remove the tumor(s) and/or the environment of the tumor(s). Thesuspension of chains of magnetosomes is administered within the cavityremaining after the chirurgical operation and the thermotherapy isstarted by applying an external magnetic field. In this case, thethermotherapy is used either to destroy part of the tumor(s), whichcould not be removed during the chirurgical operation and/or to preventthe tumor from growing again after the chirurgical operation.

In still another embodiment, a cavity is created during the chirurgicaloperation within the tumor and/or tumor environment to create a morefavourable environment for the production of heat by the chains ofmagnetosomes than that of the tumor tissue (i.e. for example a lessviscous environment), in this embodiment the chains of magnetosomes areadministered in this cavity.

The invention is also related to the use of chains of magnetosomesand/or of vesicles containing the chains of magnetosomes, as describedabove, as a mean of heating, especially of a living tissue or livingcells in vivo.

The invention is also related to the use of chains of magnetosomes(encapsulated or not in a vesicle) as a drug, especially as a drug foranti-tumoral treatment, especially for anti-tumoral heat treatment.

In one embodiment the chains of magnetosomes (encapsulated in a vesicleor not) are used as a drug, which enables the treatment of tumor(s)and/or tumor cells through a heating method.

In these uses, the vesicle may contain an active principle, which is forexample an anti-tumoral drug.

In one embodiment the vesicles containing the chains of magnetosomes andoptionally the active principle are used as a drug, which allows thetreatment of the tumor cells or of tumor via a heating method, whichinduces the release of the active principle.

In another embodiment, the chains of magnetosomes (encapsulated in avesicle or not) are used as a drug activated by a medical device, whichis the alternative magnetic field.

The invention also relates to the use of chains of magnetosomes and/orvesicles containing chains of magnetosomes and optionally an activeprinciple as a medical device, specifically designed for the treatmentof tumor(s) and/or tumor cells.

In one embodiment the chains of magnetosomes (encapsulated in a vesicleor not) are used as a medical device, which enables a magnetic treatmentof either tumors or tumor cells.

In one embodiment the chains of magnetosomes (encapsulated in a vesicleor not) are used as a medical device, which enables heating of eitherthe tumors or tumor cells or their environment.

In one embodiment the chains of magnetosomes or the vesicle containingthe chains of magnetosomes are combined with an active principle andused as a medical device, which allows the delivery of an activeprinciple within the tumors, the tumor cells or their environment.

According to a feature, use is made of a device used to induce amagnetic excitation of the chains of magnetosomes to complete themedical device.

The invention is also related to a kit containing chains of magnetosomes(encapsulated in a vesicle or not) and a device, which is able togenerate a magnetic field with the features required for a treatment ofcancer or tumors induced by heat.

In one embodiment, the vesicles belonging to the kit are used incombination with an active principle, which is encapsulated within thevesicles.

The invention will now be described in further details using thefollowing non-limiting examples.

DESCRIPTION OF THE FIGURES

FIG. 1: (a) Transmission electron microscopy (TEM) micrograph of onecell of Magnetospirillum magneticum strain AMB-1, where the arrowsindicate the localization of the chains of magnetosomes; (b) TEMmicrograph of the chains of magnetosomes extracted from the bacteria;(c) TEM micrograph of individual magnetosomes detached from the chains;(d) TEM micrograph of the chemically synthesized nanoparticles(SPION@Citrate); (e) A measurement of charge at the surface of thechains of magnetosomes (CM) and individual magnetosomes (IM) as afunction of the pH of the suspension containing these two types ofbacterial magnetosomes; (f) The infrared spectra of the chains ofmagnetosomes (CM) and individual magnetosomes (IM).

FIG. 2: (a) Variation of the temperature of a suspension of intactmagnetotactic bacterial cells as a function of time when the suspensionis subjected to an alternative magnetic field (AMF) of frequency 108 kHzand AMF amplitude of 23 mT. The lines correspond to the suspension ofcells in water (suspension) and in a 2% agarose gel (gel), respectively;(b) Same as in (a) for an AMF amplitude of 88 mT; (c) Minor hysteresisloops of the whole bacteria measured at 23 mT (squares) or 88 mT(hysteresis); (d) Specific absorption rate (SAR) of a suspension ofmagnetotactic bacteria contained either in water, or in a gel, as afunction of AMF amplitude. The hysteresis losses measured from the areaof the minor hysteresis loops of a suspension of magnetotactic bacteriacontained in a gel; (e): Plots in column bars of the SAR of the intactcells measured at 23 mT and 88 mT. The boxes represent the contributionto the observed increases in temperature due to hysteresis losses.

FIG. 3: Properties of the chains of magnetosomes extracted from cells ofMagnetospirillum magneticum strain AMB-1. (a) Increase in temperature ofthe chains of magnetosomes as a function of time in the presence of anAMF of frequency 108 kHz and AMF amplitude 23 mT. The lines indicate theheating rate of to the chains of magnetosomes suspended in water(solution) or in a 2% agarose gel (gel) respectively; (b) Same as in (a)for a AMF amplitude of 88 mT; (c) Minor hysteresis loops of chains ofmagnetosomes at 23 mT (square) and 88 mT (line); (d) SAR measured fromthe slope at 22° C. of the heating rate of the chains of magnetosomessuspended in water (suspension), or in a gel (gel), as a function of theAMF amplitude. Hysteresis losses measured from the area of the minorhysteresis loops of a suspension of chains of magnetosomes contained ina gel (hysteresis). (e): Plots in column bars of the SAR of the chainsof magnetosomes measured at 23 mT and 88 mT. Boxes represent thecontribution to the observed increases in temperature resulting fromhysteresis losses and rotation of the chains of magnetosomes,respectively.

FIG. 4: Properties of individual magnetosomes extracted from cells ofMagnetospirillumm magneticum strain AMB-1 and treated further by SDS andheat. (a) Increase in temperature of the individual magnetosomes as afunction of time when an AMF of frequency 108 kHz and amplitude 23 mT isapplied. The lines indicate the variations in temperature of theindividual magnetosomes suspended in water (suspension) or in a 2%agarose gel (gel), respectively; (b) Same as in (a) for AMF amplitude of88 mT. (c): Minor hysteresis loops of the individual magnetosomesmeasured at 23 mT (square) and 88 mT (line), (d) SAR measured from theslope at 22° C. of the heating rate of the individual magnetosomescontained either in water (suspension), or in a gel (gel), as a functionof the AMF amplitude. Hysteresis losses measured from the area of theminor hysteresis loops of the individual magnetosomes contained in a gel(hysteresis); (e): Plots in column bars of the SAR of the individualmagnetosomes measured at 23 mT and 88 mT. Boxes represent thecontributions to the observed increases in temperature due to hysteresislosses and rotation of the individual magnetosomes, respectively.

FIG. 5: (a-c): Properties of chains of magnetosomes extracted frommagnetotactic bacteria, which have been synthesized in the absence ofchelating agents and/or other transition metals than iron. Histogramsshowing the distribution in magnetosome sizes, (a), and magnetosomechain lengths, (b) of this type of magnetosomes. (c): The variation withtime of the temperature of a suspension of this type of magnetosomescontaining 406 μg/ml in maghemite when this suspension is exposed to analternative magnetic field of frequency 183 kHz and magnetic fieldstrength of either 43 mT or 80 mT. (d-f): Properties of chains ofmagnetosomes extracted from magnetotactic bacteria, which have beensynthesized in the presence of 0.4 μM EDTA. Histograms showing thedistribution in magnetosome sizes, (d), and magnetosome chain lengths,(e), of this type of magnetosomes. (f): The variation with time of thetemperature of a suspension containing this type of magnetosomes with aconcentration of 406 μg/ml of maghemite when the suspension is exposedto an alternative magnetic field of frequency 183 kHz and magnetic fieldstrength of either 43 mT or 80 mT.

FIG. 6: (a) The variation as a function of time of the temperature ofseveral suspensions containing different types of chains of magnetosomessynthesized in the presence of different chelating agents (4 μM EDTA, 4μM Rhodamine B, 4 μM Dopamine, 4 μM Alendronate) and 406 μg of maghemiteper milliliter when these suspensions are submitted to an alternativemagnetic field of frequency 183 kHz and field strength of either 43 mTor 80 mT. (b) The variation as a function of time of the temperature oftwo suspensions containing undoped and Co-doped magnetosomes organizedin chains. The concentration of these suspensions is 1.52 mg/mL and theyare exposed to an alternative magnetic field of frequency 183 kHz andfield strength of 80 mT.

FIG. 7: Properties of suspended MDA-MB-231 cells incubated in theabsence or in the presence of extracted chains of magnetosomes ofvarious concentrations (0.125 mg/mL<Cγ_(Fe203)<1 mg/mL, where Cγ_(Fe203)represents the concentration in maghemite of the suspensions) andexposed to an alternative magnetic field of frequency 183 kHz andvarious strengths (0 mT<B<60 mT, where B represents the strength of theapplied magnetic field). (a): Percentage of MDA-MB-231 living cells as afunction of the magnetic field strength for an incubation of suspensionsof chains of magnetosomes of various concentrations. (b)-(d): Variationsof temperature of suspensions containing MDA-MB-231 cells incubated inthe absence or in the presence of chains of magnetosomes of variousconcentrations (0.125 mg/mL<Cγ_(Fe203)<1 mg/mL) when an alternativemagnetic field of B=20 mT, (b), B=43 mT, (c), or B=60 mT, (d), isapplied to these suspensions.

FIG. 8: (a)-(c): Percentage of living adherent MDA-MB-231 cells as afunction of the strength of the magnetic field (0 mT<B<60 mT), which isapplied once during 20 minutes. The cells are either incubated during 24hours, D1, (a), 48 hours, D2, (b) or 72 hours, D3, (c) either in theabsence or in the presence of the extracted chains of magnetosomes ofvarious concentrations (0.125 mg/mL<Cγ_(Fe203)<1 mg/mL). (d): Percentageof living adherent MDA-MB-231 cells as a function of the magnetic fieldstrength (0 mT<B<60 mT), which is applied two times during 20 minutes.The cells are incubated during 72 hours in the absence or in thepresence of the extracted chains of magnetosomes of variousconcentrations (0.125 mg/mL<Cγ_(Fe203)<1 mg/mL).

FIG. 9: (a-c) Percentage of inhibition of MDA-MB-231 cells incubated inthe presence of four different suspensions containing chains ofmagnetosomes (CM), individual magnetosomes (IM), SPION covered bycitrate ions (SPION@Citrate), SPION covered by PEG molecules (SPION@PEG)as a function of the concentration in maghemite of these foursuspensions. (d) Percentage of cells, which become magnetic as afunction of the incubation time, when the four suspensions mentionedabove (0.125 mg/mL<Cγ_(Fe203)<1 mg/mL) are incubated in the presence ofMDA-MB-231 cells and an alternative magnetic field of 183 kHz andstrength of 43 mT is applied.

FIG. 10: (a) Experimental set-up used to treat the mice. It contains a10 kW EasyHeat power supply from Ambrell, Soultz, France, equipped witha coil of 6.7 cm in diameter where an AMF of various strengths (variedfrom 20 mT and 80 mT) is applied. A mouse is positioned inside the coilfor treatment. (b) Schematic diagram showing the heated tumor and theposition across the tumor, which is recorded during the infraredmeasurements of the temperature. (c)-(f): Study of mice treated withsuspensions of individual magnetosomes (mice 1 to 4). (c) The variationsof the tumor and rectal temperatures when the magnetic field is appliedduring the treatment. These temperatures are averaged over the differentmice treated (mice 1 to 3); (d) For a mouse showing a typical behavior,the temperature distribution measured across the treated tumor 10 minafter the treatment has started; (e) Variation of the normalized tumorvolume for the tumor in which the suspension of individual magnetosomeshas been injected (mice 1 to 3). The volumes of the tumors arenormalized by the volume of the tumor at the time of the treatment; (f)Same as in (e) for the so-called control tumor in which only PBS hasbeen injected. In mouse 4, the suspension of individual magnetosomes hasbeen injected but no magnetic field has been applied.

FIG. 11: Study of the mice treated with individual magnetosomes (mice 1to 4). (a) Photographs of the treated tumor in mouse 1 just after thetreatment (D0), 14 days after the treatment (D14) or 30 days after thetreatment (D30). (b) Micrograph of a tumor tissue collected 30 daysafter the treatment in mouse 2; (c) Enlargement of a region of (b)showing the presence of bacterial magnetosomes (blue color or darkcontrast); (d) Enlargement of a region of (c) showing magnetosomesaggregates.

FIG. 12: Study of the mice treated with suspensions containing chains ofmagnetosomes (mice 5 to 9). (a) The variations of the tumor and rectaltemperatures when the magnetic field is applied during the treatment(mice 5 to 8). The temperature is averaged over the different mice (mice5 to 8); (b) For a mouse showing a typical behavior, temperaturedistribution measured across the treated tumor 10 min after thebeginning of the treatment; (c) Evolution of the normalized tumor volumefor the tumor in which the suspension containing the chains ofmagnetosomes has been injected. The volume of the treated tumor isnormalized by the volume of the tumor at the time of the treatment; (d)Same as in (c) for the control tumor in which only PBS has beeninjected. In mouse 9, the suspension containing the chains ofmagnetosomes has been injected but no magnetic field has been applied tothe mouse.

FIG. 13: Study of the mice treated with suspensions of chains ofmagnetosomes (mice 5 to 9). (a) Photographs of the treated tumor inmouse 5 just after the treatment (D0), 14 days after the treatment(D14), 30 days after the treatment (D30). (b) Micrograph of a tumortissue collected 30 days after the treatment in mouse 5 showing thepresence of the bacterial magnetosomes (blue color or dark contrast);(c) Enlargement of (b). (d) Enlargement of (c) showing a cell with itsnucleus surrounded by bacterial magnetosomes.

FIG. 14: (a), (c), (e), (g): Variations of the tumor and rectaltemperatures when the suspensions containing the standard chains ofmagnetosomes, (a), the magnetosomes-EDTA, (c), the SPION@Citrate, (e),or the SPION@PEG, (g), are administered within the tumor and thealternative magnetic field of frequency 183 kHz and strength 43 mT isapplied during 20 minutes. The treatment is repeated 3 times with a 1day resting time between the different treatments. (b), (d), (f), (h):Variations of the normalized tumor volume (i.e. the tumor volumemeasured at day 2 to day 30 following the treatment divided by the tumorvolume measured during the day of the treatment) during the daysfollowing the treatment for the standard chains of magnetosomes, (b),the magnetosomes-EDTA, (d), the SPION@Citrate, (f), or the SPION@PEG,(h). In (b), (d), (f) and (h), the error bars are the standarddeviations estimated by taking into account the normalized tumor volumeof each mouse.

FIG. 15: (a), (c), (e), (g): Photographs of the mice, which showed thebest anti-tumoral activity 30 days after the treatment induced by heatfor the treatment carried out using the standard chains of magnetosomes,(a), the magnetosomes-EDTA, (c), the SPION@Citrate, (e), or theSPION@PEG, (g). (b), (d), (f), (h): The variations of the normalizedtumor volume during the days following the treatment for the miceshowing the best anti-tumoral activity and treated with standard chainsof magnetosomes, (b), magnetosomes-EDTA, (d), SPION@Citrate, (f) andSPION@PEG, (h).

FIG. 16: Percentage of nanoparticules in the tumor ((a), (c), (e), (g))and in the feces ((b), (d), (f), (h)) at the time of the injection (D0),3 days after the injection (D3), 6 days after the injection (D6) and 14days after the injection (D14) for an intra-tumoral administration ofsuspensions containing either chains of magnetosomes, (a), (b),individual magnetosomes, (c), (d), SPION@Citrate, (e), (f), andSPION@PEG, (g), (h).

FIG. 17: Histograms showing the magnetosome size distributions formagnetotactic bacteria synthesized in the absence of a bisphosphonicacid, (a), in the presence of 4 μM risedronate, (b), or in the presenceof 4 μM alendronate, (c). Histograms showing the magnetosome chainlength distributions for magnetotactic bacteria synthesized in theabsence of a bisphosphonic acid, (d), in the presence of 4 μMrisedronate, (e), or in the presence of 4 μM alendronate, (f).Variations of the temperatures as a function of time when an alternativemagnetic field of strength 43 mT or 80 mT is applied to a suspensioncontaining chains of magnetosomes synthesized in the absence of abisphosophonic acid, (g), in the presence of 4 μM risedronate, (h), orin the presence of 4 μM alendronate, (i).

FIG. 18: For the mouse treated with SPION, the evolution of thetemperature and tumor sizes (mice 10 to 13). (a) Evolution of the tumorand rectal temperatures during the treatment. The temperature isaveraged over the different mice treated (mice 10 to 12); (b) For amouse showing a typical behavior, temperature distribution measuredacross the treated tumor 10 min after the beginning of the treatment;(c) Evolution of the normalized tumor volume for the tumor in which thesuspension of SPION@Citrate has been injected (mice 10 to 13). Thevolume of the treated tumor is normalized by the volume of the tumor atthe time of the treatment; (d) same as in (c) for the control tumor inwhich only PBS has been injected. In mouse 14, the solution of SPION hasbeen injected but no magnetic field has been applied to the mouse.

DESCRIPTION OF THE EXAMPLES Example 1 Preparation of the Different Typesof Particles Used as Heating Sources

In this example, we describe the methods following which the differenttypes of particles used as heating sources were prepared. Theseparticles are particles contained within whole magnetotactic bacteria,chains of magnetosomes extracted from the magnetotactic bacteria,individual magnetosomes extracted from magnetotactic bacteria anddetached from the chains by heat and SDS treatment, chemicallysynthesized superparamagnetic iron oxide nanoparticles covered bycitrate ions (SPION@Citrate) or commercially available chemicallysynthesized nanoparticles covered by PEG molecules (SPION@PEG). TheSPION@PEG were purchased from the German company Micromod (Product name:Nanomag®-D-spio, Product Number: 79-00-201).

The SPION@Citrate were used as standard nanoparticles, because theypossess similar sizes than most nanoparticles used for magnetichyperthermia (See for example: Johannsen et al, European Urology 2007,52, 1653-1662 or the other references listed at the beginning of thispattern application) and a chemical coating, which stabilizes thenanoparticles but should not produce any anti-tumoral activity. TheSPION@PEG were also used as standard nanoparticles since they arecommercially available and are the same as those used by DeNardo's groupto carry out magnetic hyperthermia (See for example: De Nardo et al,Clin. Cancer Res. 2005, 11, 7087s-7092s). The efficiency of the chainsof magnetosomes in the thermotherapy was compared with that of these twostandards (SPION@Citrate and SPION@PEG).

Magnetospirillum magneticum strain AMB-1 was purchased from the ATCC(ATCC 700274). Cells were grown micro-anaerobically at room temperature(˜25° C.) in liquid culture in slightly modified revised MSGM medium(ATCC Medium 1653). In one litter, this growth medium contains 0.68 g ofmonobasic potassium phosphate, 0.85 g of sodium succinate, 0.57 g ofsodium tartrate, 0.083 g of sodium acetate, 225 of 0.2% resazurin, 0.17g of sodium nitrate, 0.04 g of L-ascorbic acid, 2 ml of a 10 mM ironquinate solution, 10 ml of Woolf's vitamins and 5 ml of Woolf'sminerals. The iron quinate solution was prepared by dissolving 0.19 g ofquinic acid and 0.29 g of FeCl₃.6H₂O in 100 milliliter of distilledwater. The solution of Woolf's minerals contained in 1 liter ofdistilled water 0.5 g of Nitrilotriacetic acid (NTA, C₆H₉NO₆), 1.5 g ofMagnesium Sulfate HEPTA (MgSO₄.7H₂O), 1 g of Sodium Chloride, 0.5 g ofmanganese sulfate (MnSO₄.H₂O), 100 mg of ferrous sulfate heptahydrate(FeSO₄.7H₂O), 100 mg of cobalt nitrate (CO(NO₃)₂.7H₂O), 100 mg ofcalcium chloride (CaCl₂), 100 mg of Zinc sulfate heptahydrate(ZnSO₄.7H₂O), 10 mg of hydrate copper sulfate (CuSO₄.5H₂O), 10 mg ofaluminium potassium sulfate dodecahydrate (AlK(SO₄).12H₂O), 10 mg ofboric acid (H₃BO₃), 10 mg of sodium molybdate (Na₂MoO₄.2H₂O), 2 mg ofsodium selenite (Na₂SeO₃), 10 mg of sodium tungstate dihydrate(Na₂WO₄.2H₂O) and 20 mg of Nickel chloride (NiCl₂.6H₂O). The solution ofWoolf's vitamins was prepared by dissolving in 1 liter of distilledwater 2.2 mg of folic acid (vitamin B9), 10.2 mg of pyridoxine (vitaminB6), 5.2 mg of Riboflavin (vitamin B2), 2.2 mg of Biotin (vitamin H orB7), 5.2 mg of thiamin (vitamin B1), 5.2 mg of nicotinic acid (vitaminB3 or PP), 5.2 mg of pantothenic acid (vitamin B5), 0.4 mg of vitaminB12, 5.2 mg of amino benzoic acid, 5.2 mg of thiotic acid and 900 mg ofpotassium phosphate. The pH of the growth medium was adjusted to 6.85using a 5M sodium hydroxide solution. Cells were harvested as describedbelow at stationary phase. Stationary phase occurred when the mediumbecame completely reduced as indicated by a change in the coloration ofthe growth medium, from pink to colorless.

Three different types of samples were prepared from intact whole cellsof M. magneticum. Cells were harvested at stationary phase bycentrifugation at 8,000 rpm for 15 min. The supernatant (spent growthmedium) was discarded and cells were resuspended in 3 ml of deionizedwater. For suspensions of whole intact cells, this sample was nottreated further. The TEM micrograph of FIG. 1( a) shows one typicalAMB-1 magnetotactic bacterium containing several chains of magnetosomes.

To extract the chains of magnetosomes, 1 ml of the cell suspension wasrecentrifuged and resuspended in 10 mM Tris.HCl buffer (pH 7.4) and thensonicated for 120 min at 30 W to lyse the cells releasing the chains ofmagnetosomes. Sonication times of 60 and 180 min were also tested andenabled to extract the chains of magnetosomes from the bacteria. For asonication time of less than 60 min, the magnetotactic bacteria were notall lysed while for a sonication time of more than 180 min, aggregationbegan to be observed due to the presence of individual aggregatedmagnetosomes.

After sonication, the suspension of chains of magnetosomes wasmagnetically separated by placing a strong magnet in neodymium (0.1-1T)next to the tube where the magnetic material was collected as a pellet.The supernate containing cells debris and other organic material wasremoved. The magnetosome chains were washed 10 times with a 10 mMTris.HCl buffer (pH 7.4) in this way and were finally resuspended insterile deionized water. A typical assembly of chains of magnetosomesextracted from the whole bacteria is shown in the TEM micrograph of FIG.1( b). The surface charge of the chains of magnetosome was measured as afunction of pH using dynamic light scattering measurements(NanoZetasizer, Malvern instruments Ltd). At physiological pH, FIG. 1(e) shows that the surface charge of the chains of magnetosomes isnegative at −22 mV. The infrared measurements were carried out using aNicolet 380 FT IR Thermo Electro. The infra-red absorption spectrum of asuspension of chains of magnetosomes was also recorded. It showed peaksarising from the functional groups carboxylic acid, amine, amide,phosphate (P—O), revealing the presence of both proteins andphospholipids within the suspension of chains of magnetosomes. Thisresult suggests that both the membrane surrounding the magnetosomes andthe filament binding the magnetosomes together are present in thissample (D. Faivre et al, Chem. Rev., 2008, 108, 4875-4898).

Individual magnetosomes (i.e. magnetosomes, which are not organized inchains) were obtained by heating the suspension of magnetosome chainsfor five hours at 90° C. in the presence of 1% sodium dodecyl sulfate(SDS) in deionized water to remove most of the biological materialsurrounding the magnetosomes, i.e. most of the magnetosome membranesurrounding the magnetosomes and the cytoskeleton responsible for thealignment of the magnetosomes in each chain (D. Faivre, Chem. Rev.,2008, 108, 4875-4898). Individual magnetosomes were washed as describedfor magnetosome chains and resuspended in deionized water. The TEMmicrograph of FIG. 1( c) shows a typical assembly of individualmagnetosomes. The individual magnetosomes possess different propertiesfrom the chains of magnetosomes. They form an aggregated assembly ofnanoparticules (FIG. 1( c)). They possess a surface charge, whichstrongly depends on their level of aggregation. When the individualmagnetosomes are sonicated and dispersed in water, they possess arelatively similar surface charge than the chains of magnetosomes at pH7. However, when they are aggregated, the individual magnetosomespossess a positive charge (10 mV at pH 7, FIG. 1( e)). The individualmagnetosomes are surrounded by phospholipid acid (presence of P-0 peakin the infra-red absorption spectrum of FIG. 1( f)), but not by proteins(absence of amide in the infra-red absorption spectrum of FIG. 1( f)),suggesting that the biomaterial, which surrounds the magnetosomes hasnot been completely removed but has been sufficiently denatured to yieldindividual magnetosomes not organized in chains.

The chemically synthesized nanoparticles (SPION@Citrate) were preparedfollowing a protocol described previously (Lalatonne et al., Phys. Rev.E, 2005, 71, 011404-1, 011404-10). To prepare non-coated γFe₂O₃particles, a solution of base (dimethylamine) was first added to anaqueous micellar solution of ferrous dodecyl sulfate (Fe(DS)₂) andmixed. The final reactant concentrations were 1.3×10⁻² mol L⁻¹ and8.5×10⁻¹ mol L⁻¹ for Fe(DS)₂ and dimethylamine, respectively. Thesolution was then stirred vigorously for 2 hours at 28.5° C. and theresulting precipitate of uncoated nanocrystals was isolated from thesupernatant by centrifugation. In the second step, the precipitate waswashed with an acidic solution (HNO₃, 10⁻² mol·L⁻¹) until a solution ofpH=2 were reached. Sodium citrate dissolved in water([Na₃C₆O₇H₅]=1.5×10⁻² mol L⁻¹) was used to coat the nanoparticles. Thesolution was subjected to sonication for 2 hours at 90° C. and theaddition of acetone induced nanocrystal precipitation. After washingwith a large excess of acetone, the precipitate was dried in air. Thenanocrystals coated with citrate ions were finally dispersed in water.The pH, which was initially ˜2, was progressively increased up to 7.4 byadding of solution of sodium hydroxide NaOH (10⁻¹ mol·L⁻¹). TheSPION@Citrate are composed of maghemite and possess a mean size of ˜10nm. A TEM micrograph of the SPION@Citrate is shown in FIG. 1( d).

The detailed properties of the SPION@PEG can be obtained from thecompany Micromod. It is indicated in the information sheet (product-No:79-00-201) provided by Micromod that the SPION@PEG possess a saturatingmagnetization of 34 emu/g, a size of about 20 nm, a polydispersity ofless than 20% and that they are stable in aqueous buffer for pH>4.

Example 2 Heat Production by Bacterial Magnetosomes Exposed to anOscillating Magnetic Field

In this example, we provide a detailed study of the mechanisms of heatproduction by magnetosomes biomineralized by magnetotactic bacteria. Thevalues of the magnetic field frequency (108 kHz) and magnetic fieldamplitude (23 to 88 mT) used to heat the different samples lie withinthe range of the magnetic field parameters used to carry out highfrequency high amplitude AMF (alternative magnetic field) hyperthermia(Ivkov et al, Clin. Cancer Res., 2005, 11, 7093s-7103s; De Nardo et al,Clin. Cancer Res., 2005, 11, 7087s-7092s; De Nardo et al, The J. Nucl.Med., 2007, 48, 437-444). For AMF hyperthermia, recommended magneticfield frequencies lie between 50 kHz and 1 MHz while the magnetic fieldamplitude needs to remain below 100 mT (Mornet et al, J. Mater. Chem.,2004, 14, 2161-2175). We compare the heat-producing properties of threedifferent types of magnetosome arrangements (Alphandéry et al, J. Phys.Chem. C, 2008, 112, 12304-12309; Alphandéry et al, ACS Nano, 2009, 3,1539-1547): 1) magnetosome chains contained within intact AMB-1magnetotactic bacteria; 2) chains of magnetosomes extracted from thebacteria that retained their magnetosome membranes; and 3) individualmagnetosome crystals whose magnetosome membranes have been mostlyremoved.

It is known that, for large ferromagnetic nanoparticles, there are twomain heat-producing mechanisms. The first one is due to the physicalrotation of magnetic nanoparticles in a magnetic field and the secondone is a result of hysteresis losses (Hergt et al, IEEE Trans. Mag.,1998, 34, 3745-3754). In order to determine which of these mechanisms isresponsible for heat production by the three different types ofmagnetosome arrangements mentioned above, we compared the heating ratesof the samples in water, in which rotation of the cells and magnetosomesis possible, with those present in a gel, where rotation is inhibited.In this way, the amount of heat generated by the rotation of thebacteria or magnetosomes and that arising from hysteresis losses can bedetermined. In order to verify that heat produced in the gel is due tohysteresis losses, we measured hysteresis losses independently usingmagnetic measurements.

Materials and Methods:

Samples were examined using a JEOL model JEM 1011 transmission electronmicroscope (JEOL Ltd., Tokyo) operating at 100 kV. Five microliters of asolution containing 2×10⁻⁴% in weight of magnetosomes were deposited ona carbon-coated copper grid and the grids were allowed to dry beforeexamination. The same relative quantity of magnetosomes were used toprepare all samples, thus aggregation in a particular sample was not aresult from a difference in the concentration of the magnetosomes.

Magnetic measurements were carried out using a vibrating samplemagnetometer (VSM, Quantum design, San Diego, Calif.). For magneticmeasurements, 25 microliters of a liquid suspension of magnetotacticbacterial cells, chains of magnetosomes or individual magnetosomescontaining 2.10⁻³% in weight of magnetosomes, were deposited on top of asilica substrate. The samples were then positioned inside a capsule madeof hard gelatin in a direction parallel to that of the magnetic field.Three types of magnetic measurements were performed, those of thesaturating isothermal remanent magnetization (SIRM) and major or minorhysteresis loops. SIRM measurements were used to determine thecomposition of the magnetosomes following a method similar to thatpreviously described (Alphandéry et al., J. Phys. Chem. C, 2008, 112,12304-12309) and showed that the magnetite in the magnetosomes had beenalmost completely oxidized to maghemite. This result was not unexpectedas our suspensions of magnetic material were not freshly prepared andmagnetite in magnetosomes has been known to oxidize to maghemite overtime (Chen et al., Earth Planet. Sci. Lett., 2005, 240, 790-802).Maghemite and magnetite have very similar magnetic properties at roomtemperature (Alphandéry et al., J. Phys. Chem. C, 2008, 112,12304-12309). The fact that the magnetosomes magnetite had transformedto maghemite does not substantially change the conclusions drawn in thispattern since maghemite and magnetite have very similar magneticproperties at room temperature (Alphandéry et al., J. Phys. Chem. C,2008, 112, 12304-12309). Major hysteresis loop measurements were carriedout at 300 K in order to determine the amount of maghemite containedwithin samples. The latter is determined by dividing the saturatingmagnetization of the samples by the saturating magnetization ofmaghemite. For nanoparticles as large as the magnetosome crystals, thesaturating magnetization is that of the bulk material (in this case bulkmaghemite). Finally, measurements of minor hysteresis loops were alsocarried out by recording the magnetization of the samples as a functionof a continuous magnetic field, which is applied between —H₀ and H₀where H₀ is 23 mT, 36 mT, 66 mT or 88 mT.

These experiments were carried out with the whole bacteria, chains ofmagnetosomes and individual magnetosomes either suspended in ultrapuredeionized water (18.6 MΩ) or in aqueous agarose gel (2% by weight). Theconcentration of maghemite was 457 μg ml⁻¹ for the liquid suspensioncontaining the whole cells, 435 μg ml⁻¹ for that containing the chainsof magnetosomes and 380 μg ml⁻¹ for that containing the individualmagnetosomes. 250 μl of each of these three suspensions were pouredinside polypropylene tubes and positioned at the center of a coilproducing an oscillating magnetic field of frequency 108 kHz, the fieldamplitude being fixed at 23 mT, 36 mT, 66 mT or 88 mT. In order togenerate the alternating current, the coil was connected to a generator(Celes inductor C97104) and the temperature was measured using anoptical fiber probe (Luxtron STF-2, BFi OPTiLAS SAS).

Results and Discussions:

FIG. 1( a) depicts a transmission electron micrograph (TEM) of cells ofMagnetospirillum magneticum strain AMB-1 showing typical chains ofmagnetosomes. The volume occupied by magnetosomes in a whole cell israther small, typically ˜0.02%. An aqueous suspension containing intactwhole cells of M. magneticum was subjected to an oscillating magneticfield of frequency ν=108 kHz and field amplitudes of H₀=23 mT and H₀=88mT. The heating rates of cells suspended in liquid increased when themagnetic field strength was increased from 23 mT to 88 mT (FIGS. 2( a)and 2(b)). From the slopes of the variations with time of thetemperature measured at 22° C. (ΔT/δT), we estimated the SAR of theintact cells suspended in liquid using the Equation 1 below (Mornet etal., J. Mater. Chem., 2004, 14, 2161-2175; Hergt et al., J. Magn. Magn.Matter., 2005, 293, 80-86):

$\begin{matrix}{{SAR} = {{C_{water}\left( \frac{\Delta \; T}{\delta \; t} \right)}\frac{1}{x_{m}}}} & (1)\end{matrix}$

where C_(water) is the specific heat capacity of water (C_(water)=4.184J/g·K) and x_(m) is the concentration of iron in g per ml of solvent(water). Using the above formula, we deduced that the SAR of the wholebacterial suspension increased from 108±32 W/g_(Fe) to 864±130 W/g_(Fe)when the magnetic field amplitude was increased from 23 mT to 88 mT. Inorder to determine if the amount of heat (SAR) generated by the wholemagnetotactic bacteria arises from the rotation of the whole bacteria,from hysteresis losses or from both of these mechanisms, we measured theareas of the minor hysteresis loops of the whole intact cells (FIG. 2(c)), which provide estimates of the hysteresis losses of the wholecells. Using the method of Hergt et al (Hergt et al., J. Magn. Magn.Matter., 2005, 293, 80-86) we deduced from the areas of the minorhysteresis loops shown in FIG. 2( c) that the hysteresis losses of theintact cells increased from 54±25 W/g_(Fe) at 23 mT to 810±121 W/g_(Fe)at 88 mT. These SAR values are similar to those measured for thebacterial cells in suspension (FIG. (2 d)). Unexpectedly, the SARdetermined for cells fixed in agarose gel, was significantly smallerthan the areas of the minor hysteresis loops and did not seem to providegood estimates of hysteresis losses (FIG. 2( d)). This might be due toloss of some of the bacterial cells during the preparation of the gelyielding a lower concentration of magnetosomes than in the othersamples. From these results, we conclude that the rotation of intactbacterial cells does not contribute to the production of heat in thiscase. Due to their large weight and volume, intact cells of M.magneticum are not able to rotate sufficiently well under theapplication of an external magnetic field to generate heat. The absenceof contribution of the rotation can be confirmed by estimating the SARdue to the rotation of the bacterial cells, SAR_(rot). The latter isestimated using Equation 2 below (Hergt et al., IEEE Trans. Mag. 1998,34, 3745-3754).

$\begin{matrix}{{SAR}_{rot} = {\frac{1}{2}\frac{\left( {M_{s}H_{o}V} \right)^{2}}{K_{b}T}\frac{1}{\rho \; V}\frac{1}{\tau_{b}}\frac{\left( {\omega\tau}_{b} \right)^{2}}{1 + \left( {\omega\tau}_{b} \right)^{2}}}} & (2)\end{matrix}$

In (2), we have assumed that the Brownian relaxation time, τ_(b), ismuch smaller than the Néel relaxation time τ_(n), where τ_(b)=3ηV/K_(b)Tand τ_(n)=τ₀exp(E_(a)/K_(b)T). For the different samples, τ_(b) liesbetween 2.5 10⁻⁵ sec. and 0.3 sec (Mornet et al, J. Mater. Chem., 2004,14, 2161-2175. Given that τ₀˜10⁻⁹ sec and the ratio between theanisotropy energy of a chain of magnetosomes and the thermal energy,E_(a)/K_(b)T˜480 (Alphandéry et al., ACS Nano 2009, 3, 1539-1547), wefind that τ_(n)˜3. 10³⁸ sec. and hence τ_(b)/τ_(n)<<1. This justifiesthe use of (2) to measure the SAR. In Equation 2, ω=2πf, where f=108 kHzis the frequency of the oscillating magnetic field, M_(s) is thesaturating magnetization of maghemite (M_(s)=390 emu/cm³), H₀ is theamplitude of the applied magnetic field (23 mT<H₀<88 mT), V˜20 10⁻¹⁷ cm³is the volume of a typical chain of magnetosomes (Alphandéry et al., J.Phys. Chem. C, 2008, 112, 12304-12309), ρ˜5 g/cm³ is the specific weightof maghemite, K_(b)˜1.38 10⁻²³ J/K is the Boltzmann constant andτ_(b)˜10 sec is the Brownian relaxation time of an intact bacterial cellin water. The Brownian relaxation times are estimated using the formulaτ_(b)=3ηV_(h)/K_(b)T, where V_(h) is the hydrodynamic volume. For thewhole magnetotactic bacteria, we consider that V_(h)=4/3πr³, where r ishalf the typical length of a bacterium (1.5 μm). Using these values, wefind that SAR_(rot) lies between 5.10⁻² W/g_(Fe) and 7.10⁻¹ W/g_(Fe) forH₀ values between 23 and 88 mT. These values are much smaller than themeasured SAR due to hysteresis losses, which are ˜82±58 W/g_(Fe) at 23mT and ˜841±153 W/g_(F), at 88 mT (FIG. 2( d)), where these values areaverages between the SAR deduced from the heating rate in solution andthose deduced from the measurements of the minor hysteresis loops. Thus,rotation of the whole bacterial cells does not appear to contribute tothe observed increase in temperature. As indicated in FIG. 2( d), theSAR appears to be completely due to hysteresis losses. These lossesbecome much more significant at higher magnetic field amplitudes (SAR˜841±153 W/g_(Fe) at 88 mT) than at 23 mT (SAR ˜82±58 W/g_(Fe))). Thisfinding, increased hysteresis losses with increasing magnetic fieldamplitude, has been previously observed for chemically-synthesizedmagnetite nanoparticles (Hergt et al., IEEE Trans. Mag., 1998, 34,3745-3754). The SAR per cycle of the whole cells in suspension, which isdefined as the SAR divided by the frequency of the oscillating magneticfield, lies between 0.7±0.5 J/kg_(Fe) and 7.8±1.4 J/kg_(Fe). Thesevalues are higher than most of those obtained withchemically-synthesized magnetic nanoparticles, which typically liebetween 0.001 J/kg_(Fe) and 1.2 J/kg_(Fe) for a wide range of magneticnanoparticle sizes and compositions as well as for a large choice ofmagnetic field frequencies and amplitudes (Dutz et al, J. Magn. Magn.Mater., 2007, 308, 305-312; Ma et al., J. Magn. Magn. Mater., 2004, 268,33-39; Jordan et al., J. Nano. Res., 2003, 5, 597-600; Brusentsov etal., J. Magn. Magn. Mater., 2001, 225, 113-117; Chan et al., Scientificand clinical applications of magnetic carriers, Häfeli et al. (eds.),Plenum Pres, NY, 1997, 607-618). We conclude that the suspensions ofwhole magnetotactic bacteria produce a larger amount of heat than mostof the chemically-synthesized magnetic nanoparticles under ourexperimental conditions.

Chains of magnetosomes were extracted from bacterial cells to presumablyenhance their rotation in the magnetic field without the cell structureinterfering with rotation. To verify that the magnetosomes were actuallyextracted from the bacteria and that they remain as chains, we usedelectron microscopy. FIG. 1( b) shows typical assemblies of chains ofmagnetosomes (Alphandéry et al., ACS Nano., 2009, 3, 1539-1547;Alphandéry et al., J. Phys. Chem. C, 2008, 112, 12304-12309), which donot aggregate into clumps but are sufficiently close one to another aschains to be interacting magnetically. Heat production rates of thechains of magnetosomes are shown in FIGS. 3( a) and 3(b) for themagnetic field amplitudes of 23 mT and 88 mT, respectively. In solution,they are characterized by a ˜43° C. increase over a time period of 1500sec. at 23 mT (FIG. 3( a)) and by a ˜48° C. increase over the same timeperiod at 88 mT (FIG. 3( b)). These heating rates are between about 2and about 10 times larger than those obtained with the whole cells(FIGS. 2( a), 2(b), 4(a) and 4(b)). This suggests either that the chainsof magnetosomes produce larger hysteresis losses than intact bacteriacells or that their rotation in the oscillating magnetic contributes toheat production or both. In order to discern which, if any, of theseexplanations is responsible for the greater heat production rates,hysteresis losses of the chains of magnetosomes were determined. FIG. 3(c) shows the minor hysteresis loops of the chains at 23 mT and 88 mT.The areas of the minor hysteresis loops for the chains of magnetosomewere less than those obtained with the intact bacteria cells (FIG. 2(c)). This decrease is likely due to magnetic interactions between thechains of magnetosomes (Alphandéry et al., J. Phys. Chem. C, 2008, 112,12304-12309) and thus we conclude that the higher heat production rateobserved for the chains of magnetosomes compared to the intact bacterialcells suspended in liquid is not due to an increase of hysteresis lossesbut to the rotation of the chains. The contribution of the rotation toheat production by the chains of magnetosomes can be further confirmedby estimating the SAR of the chains of magnetosomes suspended in liquid.Using equation (1) and the values of ΔT/δT for the chains ofmagnetosomes in suspension (FIGS. 3( a) and 3(b)), we find that the SARincreases from ˜864±86 W/g_(Fe) at 23 mT to ˜1242±124 W/g_(Fe) at 88 mT(FIG. 3( d)). These SAR values are larger than the hysteresis lossesdeduced either from the SAR of the chains of magnetosomes in the gel(SAR ˜54±22 W/g_(Fe) at 23 mT and SAR ˜487±97 W/g_(Fe) at 88 mT) or fromthe areas of the minor hysteresis loops (SAR ˜108±41 W/g_(Fe) at 23 mTand SAR ˜486±97 W/g_(Fe) at 88 mT). In order to confirm the contributionof rotation to the heat-producing mechanism of the chains ofmagnetosomes, we determined SAR_(rot) using Equation 2, which factors inthe Brownian relaxation time. This formula is only applicable below thesaturating region where the SAR shows a strong dependence on the fieldamplitude (Hergt et al., IEEE Trans. Mag., 1998, 34, 3745-3754). Sincesaturation occurs above ˜36 mT (FIG. 3( d)), we only measured the SAR at23 mT. Using a Brownian relaxation time τ_(B)˜1.2 10⁻⁴ sec, we find thatSAR_(rot)˜3600 W/g_(Fe) at 23 mT which is larger than the SAR of 774±145W/g_(Fe) we measured experimentally by measuring the difference betweenthe SAR of magnetosome chains suspended in liquid (864±86 W/g_(Fe)) andthe SAR due to the hysteresis losses (90±59 W/g_(Fe)). The differencebetween the theoretical prediction and the experimental observation maybe explained by the partial aggregation of the chains of magnetosomes.We conclude that the rotation contributes to the heating mechanism ofthe chains of magnetosomes and that this contribution decreases from90±10% of the SAR at 23 mT down to 40±10% of the same SAR at 88 mT (FIG.3( c)). This decrease could be explained by the stronger enhancementwith increasing magnetic field amplitude of the hysteresis losses thanof the SAR due to the rotation of the chains of magnetosomes in themagnetic field (Hergt et al., IEEE Trans. Mag. 1998, 34, 3745-3754).

The last sample we tested was a suspension of individual magnetosomeswhose membranes had been mostly removed using a combination of heat anda detergent that dissolved lipids, sodium dodecyl sulfate (SDS). Thesecrystals do not remain in chains as shown in FIG. 1( c). Thesenanocrystals interact and organize within compact assemblies ofindividual nanocrystals (Alphandéry et al., ACS Nano., 2009, 3,1539-1547, Alphandéry et al., J. Phys. Chem., 2008, 112, 12304-12309;Kobayashi et al., Earth Planet. Sci. Lett., 2006, 245, 538-555) unlikethe magnetosomes with membranes shown in FIG. 1( b). The heating ratesof the liquid suspension containing these individual magnetosomes areshown in FIGS. 4( a) and 4(b) for the magnetic field amplitudes of 23 mTand 88 mT. They are lower than those observed for the chains ofmagnetosomes suspended in liquid both at 23 mT and 88 mT (FIGS. 3( a),3(b), 4(a) and 4(b)). The difference in solution heating rates observedbetween the chains of magnetosomes and the individual magnetosomes caneither be due to a difference in the contribution of the magnetosomerotation or hysteresis losses to the SAR or a combination of both. Thehysteresis losses were estimated either from the areas of the minorhysteresis loop (FIG. 4( c)) yielding SAR values lying between 270±100W/g_(Fe) at 23 mT and 427±85 W/g_(Fe) at 88 mT or from the heating ratesof the individual magnetosomes in the gel (FIGS. 4( a) and 4(b))yielding SAR values lying between 135±70 W/g_(Fe) at 23 mT and 432±86W/g_(Fe) at 88 mT. The hysteresis losses estimated by either of the twomethods mentioned above (FIG. 4( d)) are similar to those estimated forthe chains of magnetosomes (FIG. 3( d)). Therefore, the difference inSAR observed between the chains of magnetosomes and the individualmagnetosomes suspended in liquid must result from a difference in theability of the structures to rotate in the magnetic field. Equation 2predicts that the SAR due to the rotation of the individual magnetosomessuspended in liquid should be the same as that deduced for the chains ofmagnetosomes, SAR_(rot)˜3600 W/g_(Fe) at 23 mT. Therefore the lowerheating rate observed for the individual magnetosomes is most likely dueto the fact that the latter are more prone to aggregation into clumpsthan the chains of magnetosomes. Aggregation of the individualmagnetosomes is clearly evident using electron microscopy (FIG. 1( c))and can also be observed visually in liquid suspension. It preventsthese magnetosomes from rotating as easily as the chains ofmagnetosomes.

From these results, we can draw the following conclusions:

-   (i) The SAR of each of the three magnetic samples (whole    magnetotactic bacteria, chains of magnetosomes and individual    magnetosomes) is larger than that reported for smaller    superparamagnetic nanoparticles.-   (ii) The predominant contribution to heat production by the intact    bacterial cells appears to be hysteresis losses while physical    rotation and hysteresis losses are both responsible for the    generation of heat for the chains of magnetosomes and individual    magnetosomes mixed in solution.-   (iii) By contrast to their behavior in solution, the chains of    magnetosomes and individual magnetosomes should less be able to    rotate in vivo. Therefore the amount of heat that they should    generate in vivo could be predicted by measuring their hysteresis    losses. Since the chains of magnetosomes and individual magnetosomes    have similar hysteresis losses, they presumably are both    equivalently good candidates for the in vivo heat therapy.

Example 3 Improved Heating Efficiency of Extracted Chains ofMagnetosomes Obtained by Synthesizing the Magnetotactic Bacteria in thePresence of Various Chelating Agents and/or Transition Metals

In this example, we describe various methods to improve the heatingefficiency of the extracted chains of magnetosomes suspended in water.These methods use various additives introduced within the growth mediumof AMB-1 magnetotactic bacteria. These additives are chelating agentssuch as bisphosphonate molecules, dopamine, rhodamine, EDTA ortransition metals such as cobalt.

Materials and Methods:

The growth medium of the magnetotactic bacteria was first prepared byfollowing the same method as that described in example 1. Then one ofthe following additives was added to the growth medium of themagnetotactic bacteria: 0.4 μM, 4 μM or 40 μM of different types ofbisphosphonic acids (alendronate, risedronate or neridronate), 4 μM, 20μM or 400 μM of a solution of rhodamine, 0.4 μM or 4 μM of a solution ofEDTA, 0.4 μM, 4 μM or 40 μM of a solution of dopamine, 2 μM or 20 μM ofa solution of cobalt quinate. 1 mL of a suspension of magnetotacticbacteria was inserted within one litter of the above growth media andthe bacteria grew during 10 days. After 10 days of growth, the bacteriawere collected and the chains of bacterial magnetosomes were extractedfrom the bacteria following the same protocol as that described in theexample 1. Five microliters of a suspension of chains of bacterialmagnetosomes, containing 2×10⁻⁴% in weight of magnetosomes were thendeposited on top of a carbon grid for transmission electron microscopy(TEM) analysis. TEM was used to determine the sizes of the magnetosomesand to estimate the lengths of the chains. In order to evaluate theheating properties of the various types of extracted chains ofmagnetosomes, the latter were mixed in water. The concentrations of thedifferent suspensions were estimated as the quantity of maghemite permilliliter. They were 0.3 mg/mL for the suspension containing themagnetosomes synthesized in the presence of several bisphosphonic acids,1.52 mg/mL for the suspensions containing Co-doped magnetosomes and0.406 mg/mL for that containing the magnetosomes synthesized in thepresence of EDTA, rhodamine, dopamine or alendronate. The suspensionswere heated under the application of an alternative magnetic field offrequency 183 kHz and strengths of 43 mT or 80 mT. The variation oftemperature of these suspensions was measured using a thermocouplemicroprobe (IT-18, Physitemp, Clifton, USA).

Results and Discussion:

In this section, we compare the properties of chains of magnetosomes,which have been obtained by cultivating the magnetotactic bacteria inthe standard conditions, i.e. in the absence of chelating agents and/ortransition metals (CM-Control) with those of the magnetosomes, whichhave been obtained by cultivating the magnetotactic bacteria in thepresence of 0.4 μM EDTA (CM-EDTA). The results of the CM-EDTA arepresented since they result in the most important change of themagnetosome properties, i.e. the largest increase in the magnetosomesizes, magnetosome chain lengths and heating efficiency compared withthe CM-Control.

As shown in the histograms of FIGS. 5( a) and 5(d), both for theCM-Control and the CM-EDTA, the magnetosome size distributions seem tobe bimodal with a higher percentage of large than small magnetosomes.The percentage of large magnetosomes is higher for the CM-EDTA (FIG. 5(d)) than for the CM-Control (FIG. 5( a)). Moreover, the fits of themagnetosome size distributions indicate that the size of the largemagnetosomes increases from ˜42 nm for the CM-Control (FIG. 5( a)) up to˜60 nm for the CM-EDTA (FIG. 5( d)). We also observe that the percentageof small magnetosomes (<30 nm) is significant for the CM-Control (>25%,FIG. 5( a)) while it is small for the CM-EDTA (<10%, FIG. 5( d)). As canbe observed by comparing the histograms presented in FIGS. 5( b) and5(e), the average magnetosome chain length also increases from ˜150 nmfor the CM-Control (FIG. 5( b)) up to ˜300 nm for the CM-EDTA (FIG. 5(e)). Long chains of magnetosomes (>800 nm in length) are only presentfor the CM-EDTA (FIG. 5( e)). When the alternative magnetic field isapplied to the suspensions containing the CM-EDTA, it produces anincrease in temperature, which is larger for the CM-EDTA than for theCM-Control for both magnetic field strengths of 43 mT and 80 mT (FIGS.5( c) and 5(f)). Moreover, the saturating temperatures for the CM-EDTA(35° C. and at 43 mT and 45° C. and at 80 mT, FIG. 5( f)) are bothhigher than those obtained for the CM-Control (28° C. at 43 mT and 35°C. at 80 mT, FIG. 5( c)). These features reveal the higher heatingcapacity of the CM-EDTA as compared with the CM-Control, which could beattributed to an increase in the magnetosome sizes and/or magnetosomechain lengths.

For a series of other chelating agents introduced in the bacterialgrowth medium, the same trends as those observed with 0.4 μM EDTA can beobserved but with a less pronounced effect. As shown in FIG. 6( a), thetemperature increases more rapidly under the application of analternative magnetic field of strength 43 mT or 80 mT for the chains ofmagnetosomes issued from the bacteria cultivated in the presence ofvarious chelating agents (4 μM Rhodamine B, 4 μM dopamine, 4 μMAlendronate) than for the CM-control.

When the magnetosomes were synthesized in the presence of 4 μMrisedronate or 4 μM alendronate, the percentage of magnetosome withsizes larger than 45 nm becomes larger than that of the magnetosomessynthesized in the absence of bisphosphonic acid (FIGS. 17( a), 17(b)and 17(c)). The percentage of chains with lengths larger than 400 nm isalso higher for the magnetosomes synthesized in the presence ofbisphosphonic acid (FIGS. 17( e) and 17(f)) than for those synthesizedin the absence of bisphosphonic acid (FIG. 17( d)). Because of thesebehaviors, the variation of temperature induced by the application of amagnetic field is larger for the magnetosome synthesized in the presenceof 4 μM risedronate (FIG. 17( h)) or 4 μM alendronate (FIG. 17( i)) thanfor those synthesized in the absence of bisphosphonic acid (FIG. 17(g)). This behavior is observed both for a magnetic field strength of 40mT and 80 mT. For bisphosphonic acids with concentrations of 0.4 μMintroduced in the growth medium, similar results as those obtained for aconcentration of 4 μM were observed. By contrast, for a concentration inbisphosphonic acid of 40 μM introduced in the bacterial growth medium,the properties of the chains of bacterial magnetosomes were notsignificantly different from those of the bacterial magnetosomessynthesized in the standard conditions. These results suggest that theconcentrations in bisphosphonic acid necessary to reach optimum heatingefficiency lie between 0.1 and 40 μM, in particular between 0.1 and 10μM, typically between 0.4 and 4 μM. A third bisphosphonic acid(neridronic acid) was also tested and yielded similar results as thoseobtained with alendronic or risedronic acid.

The AMB-1 magnetotactic bacteria were also cultivated in a growthmedium, which contained the chemicals of ATCC Medium 1653 and a 20 μM or400 μM solution of rhodamine. When 55 μg of chains of magnetosomessynthesized in the presence of rhodamine and mixed in one milliliter ofwater were subjected to an alternative magnetic field of 43 mT thetemperature of the suspension increased by 3 degrees in 30 minutes. Forthe chains of magnetosomes synthesized in the absence of rhodamine, atemperature increase of only one degree was observed in the sameexperimental conditions. This shows that the presence of rhodamine inthe growth medium yields improved heating capacity of the chains ofmagnetosomes.

The heating efficiency of the extracted chains of magnetosomessynthesized by introducing a 20 μM cobalt quinate solution within thebacterial growth medium has also been tested. The presence of cobaltwithin some of the magnetosomes has been detected using energy electronloss spectroscopy (EELS) measurements. This result agrees with that ofStaniland et al (S. Staniland et al, Nature Nanotech., 2008, 3,158-162), which also showed the presence of cobalt within themagnetosomes for magnetotactic bacteria synthesized in similarconditions. As shown in FIG. 6( b), when the suspension containing theCo-doped magnetosomes (C_(γFe203)=1.52 mg/mL) is exposed to analternative magnetic field of strength 80 mT and frequency 183 kHz, thetemperature of the suspension increases more than that containing theCM-Control. Since the magnetosome sizes and magnetosome chain lengthshave been shown to be very similar for the undoped and Co-dopedmagnetosomes, the enhanced heating efficiency of the Co-dopedmagnetosomes could be explained by an increase in theirmagnetocrystalline anisotropy.

From these results, we can draw the following conclusions:

-   (i) The introduction of iron chelating agents of concentrations    lying between 0.1 μM and 1 mM within the AMB-1 bacterial growth    medium yields improved heating properties of the extracted chains of    magnetosomes mixed in solution. We believe that this behavior is due    to an increase of the magnetosome sizes and/or magnetosome chain    lengths when the bacteria are cultivated in these conditions.-   (ii) The introduction of cobalt quinate with a concentration lying    between 0.1 μM and 1 mM within the AMB-1 bacterial growth medium    also yields improved heating properties of the extracted chains of    magnetosomes mixed in solution. We believe this behavior is due to    an increase of the magnetocrystalline anisotropy of the magnetosomes    doped with cobalt.-   (iii) The introduction of iron chelating agents and/or cobalt    quinate within the bacterial growth medium provides a way to enhance    the heating efficiency of the chains of magnetosomes. This open the    way to use these chains of magnetosomes in a smaller amount in the    thermotherapy, hence reducing the risk of toxicity induced by the    presence of the chains of magnetosomes.

Example 4 Efficiency of the Thermotherapy Evaluated In Vitro Materialsand Methods:

-   MDA-MB-231 cells were obtained from the American Type Culture    Collections (ATCC). The cells lines were cultivated in Dulbecco's    modified Eagle's medium (DMEM) supplement, which contained 10% fetal    calf serum (FCS), 2 mM I-glutamine, 1 mM sodium pyruvate, 50 U/ml    streptomycin (all purchased from Life Technologies Inc.). All in    vitro experiments were carried out at 37° C. in an incubator with 5%    of CO₂.

Cell viability was evaluated using the so-called MTT (microculturetetrazolium assay, T. Mosmann, 1983, J. Immunol. Methods, 65, 55-63).This technique measures the ability of mitochondrial enzymes to reduce3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (purchasedfrom Sigma, St Louis, Mo., USA) into purple formazan crystals. Cellswere seeded at a density of 2 10⁴ cells per well in 96-well flat-bottomplates (Falcon, Strasbourg, France) and incubated within the culturemedium during 24 hours. Then, the medium was removed and replaced by 10%FCS-medium containing the various nanoparticles (chains of magnetosomes,individual magnetosomes, SPION@Citrate and SPION@PEG) with differentconcentrations in maghemite (0.125 mg/mL<C_(γFe203)<1 mg/mL). Thesesuspensions were exposed (or not for the control) to an alternativemagnetic field of frequency 183 kHz and strength of 43 mT. The treatmentwas carried out during 20 minutes either one time or two times. After 72hours of incubation, the cells were washed with a phosphate buffersaline (PBS from Life Technologies) and incubated with 0.1 mL of MTT (2mg/mL) for an additional 4 hours at 37° C. The insoluble product(composed essentially of formazan) was then dissolved by adding 100 μlof DMSO (Sigma-Aldrich). The absorbance of the solubilized formazan wasmeasured at 570 nm using a Labsustem Multiscan MS microplate reader. Itprovided an estimate of the number of functional mitochondria, a number,which is proportional to the number of living cells. The percentage ofinhibition was then estimated as the number of dead cells (i.e. cells inapotosis) divided by the total number of cells.

For the toxicity studies, the cells were seeded on Petri dishes(diameter of 30 nm with 50 000 cells per Petri dish), and grew during 24hours. After this initial period of growth, the cells were incubated inthe presence (or not for the control) of the various types ofnanoparticles studied during 24 hours, 48 hours or 72 hours. At the endof the incubation time, the cells were exposed (or not for the control)to an alternative magnetic field of frequency 183 kHz and strengths 20mT, 43 mT or 60 mT. The treatment was carried out during 20 minuteseither one time or two times. Following the treatment, the cells werewashed twice with PBS. Then in order to harvest the cells, 250 μl ofTrypsin-EDTA were added to the adherent cells. 750 μl of the liquidmedium were added to the harvested cells to homogenize the suspension.The suspension was then centrifugated at 700 G during 3 minutes, thesupernate was removed and the cells were resuspended in 1 mL of PBS. Inorder to evaluate the percentage of living cells, 5 μl of propidiumiodide (PI) (1 mg/mL mixed in ethanol, Sigma Aldrich) was added to thecell suspensions. Since PI only penetrates within dead cells, themeasurement of its fluorescence provides an estimate of the percentageof dead cells. From this estimate, we could deduce the percentage ofliving cells. In order to measure the fluorescence of PI, the cells wereanalyzed in a flow cytometer (Beckton Dickinson FACSCalibur 3C), whichcontains an argon laser with an emission at 488 nm and a detector FL3-Hable to detect the fluorescence of PI excited by the laser. Ten thousandcells per sample were measured to determine the percentage of livingcells.

In order to measure the heating properties of the cell suspensions invitro, essentially the same experiment as that described above for theadherent cells has been carried out for the cells in suspension. Theonly difference in this case is that the cells have immediately beenmixed with the chains of magnetosomes and treated by application of themagnetic field. The temperature was measured with the thermocouplemicroprobe (IT-18, Physitemp, Clifton, USA), which measures thetemperature macroscopically (i.e. the temperature of the cell suspensionas a whole but not the temperature within each individual cell).

In order to estimate the number of magnetic cells, essentially the sameprotocol as that described above for the cells in suspension wasfollowed. 50 000 cells contained within the liquid medium describedabove were incubated in the presence of the various nanoparticles during5 to 20 minutes. During the incubation, an alternative magnetic field offrequency 183 kHz and field strength of 43 mT was applied. Aftertreatment, the magnetic cells were collected by positioning a strongmagnet of 0.6 mT close to the cells in suspension. The supernatecontaining the non-magnetic cells was removed while the cells which hadbeen attracted by the magnet were resuspended in 1 mL of PBS. Thepercentage of magnetic cells was then estimated using the flowcytometer.

Results and Discussions:

For the treatment with the cells in suspension, the cells were firstincubated during a few minutes in the presence of a suspension of chainsof magnetosomes of various concentrations. At the same time, analternative magnetic field of frequency 183 kHz and various strengths (0mT<B<60 mT) was applied. The percentage of living cells was thenmeasured in the flow cytometer for the different magnetic fieldstrengths. FIG. 7( a) shows that the percentage of living cells is high(>80%) for the different strengths of the applied magnetic field,indicating low toxicity. This could be explained by the fact that thecells don't reach the state of apoptosis just after the treatment. Forthe cells incubated in the presence of various quantities of chains ofmagnetosomes during a few minutes, the variations of temperature of thesuspensions, which is due to the application of the alternative magneticfield, are shown in FIGS. 7( b), 7(c) and 7(d) for magnetic fieldstrengths of 20 mT (FIG. 7( b)), 43 mT (FIGS. 7( c)) and 60 mT (FIG. 7(d)) respectively. As shown in FIG. 7( b), the magnetic field strength of20 mT is too low to result in an increase of temperature. By contrast,FIG. 7( d) shows that the magnetic field strength of 60 mT induces alarge increase in temperature. The latter takes place even for the cellsincubated in the absence of the chains of magnetosomes indicating thatit arises from the Foucault's currents. The magnetic field of strength43 mT is the one which provides an acceptable behavior, i.e. novariation of temperature in the absence of the chains of magnetosomesand an increase in temperature, which increases with an increasingquantity of chains of magnetosomes incubated (FIG. 7( c)).

For the adherent cells incubated during more than a few minutes, thepercentage of living MDA-MB-231 cells has also been measured as afunction of magnetic field strengths (FIG. 8). The cells have beenincubated in the presence of suspensions of chains of magnetosomes ofvarious concentrations during 24 hours (D1, FIG. 8( a)), 48 hours (D2,FIG. 8( b)) or 72 hours (D3, FIGS. 8( c) and 8(d)). The treatmentinduced by heat has been carried out either one time (FIGS. 8( a) to8(c)) or two times (FIG. 8( d)). In the absence of the magnetic field,the presence of the chains of magnetosomes is toxic (less than 50% ofliving cells) for 1 mg of chains of magnetosomes incubated during 48hours or 72 hours. For all the other conditions tested, the presence ofthe chains of magnetosomes exhibits low toxicity (more than 50% ofliving cells). In the presence of a magnetic field, FIGS. 8( a) to 8(c)show that the percentage of living cells decreases significantly for amagnetic field of 43 mT or more and for a quantity of magnetosomesincubated of more than 0.5 mg. FIG. 8( d) shows that by repeating thetreatment twice it is possible to improve the efficiency of thetreatment, where the latter is defined by a high percentage of livingcells destroyed using a small quantity of magnetosomes. Indeed, for atreatment carried out two times, the percentage of living cells of 20%is reached for 0.25 mg of chains of magnetosomes used (B=43 mT) (FIG. 8(d)) compared with 0.5 mg for a treatment carried out one time (B=43 mT)(FIG. 8( c)).

The percentage of inhibition of MDA-MB-31 cells incubated in thepresence of the various types of nanoparticles mentioned above has alsobeen estimated either in the absence of a magnetic field (FIG. 9( a)) orin the presence of a magnetic field of 43 mT for a treatment carried outeither one time (FIG. 9( b)) or two times (FIG. 9( c)). In allconditions tested, the percentage of inhibitions of the cells is largerfor the MDA-MB-31 cells incubated in the presence of the chains ofmagnetosomes than for those incubated in the presence of all other typesof nanoparticles (the individual magnetosomes, the SPION@Citrate and theSPION@PEG). The best conditions for the treatment (i.e. those, whichresult in a high percentage of inhibition in the presence of a magneticfield and in a low percentage of inhibition in the absence of a magneticfield) are obtained for the smallest quantity of chains of magnetosomesof 0.125 mg used and for the treatment carried out more than one time(FIGS. 9( a) and 9(c)).

FIG. 9( d) shows the percentage of MDA-MD-231 cells, which becomemagnetic when they are incubated in the presence of the various types ofnanoparticles while the alternative magnetic field of 43 mT is appliedbetween 0 and 20 minutes. The percentage of magnetic cells is high forthe cells incubated in the presence of the chains of magnetosomes and ofthe SPION@Citrate. It lies between 40% and 90% depending on how long thealternative magnetic field is applied (FIG. 9( d)). FIG. 9( d) alsoshows that the percentage of internalization of the individualmagnetosomes within the MDA-MD-231 cells is low (<20%). This may beexplained by the tendency of the individual magnetosomes to aggregate,which prevents them from penetrating within the cells As shown in FIG.9( d), the SPION@PEG possess a very low percentage of internalizationwithin the MDA-MD-231 cells, indicating that the percentage ofinternalization of magnetic nanoparticles within eukaryotic cells afterapplication of an alternative magnetic is strongly dependent on the typeof nanoparticles used.

From these results, we can draw the following conclusions:

-   (i) In the absence of treatment, the cytotoxicity of the chains of    magnetosomes is low for a quantity of chains of magnetosomes below 1    mg.-   (ii) The magnetic field strength of 43 mT yields the best heating    property for MDA-MB-231 cells suspended in the presence of chains of    magnetosomes of various concentrations.-   (iii) The best conditions are reached for the lowest quantity of    chains of magnetosomes incubated (0.125 mg) and for the treatment    repeated twice.-   (iv) The higher percentage of inhibition reached for the chains of    magnetosomes as compared with the individual magnetosomes could be    due to a better internalization of the chains of magnetosomes within    the MDA-MD-231 cells as compared with that of the individual    magnetosomes.-   (v) The higher percentage of inhibition observed for the cells    incubated in the presence of the chains of magnetosomes compared    with that observed for the cells incubated in the presence of the    SPION@Citrate may be explained either by the higher SAR of the    chains of magnetosomes or by the more homogenous heating of the    chains of magnetosomes or by a combination of both of these    properties.

Example 5 Heating Efficiency and Antitumoral Activity of VariousBacterial Magnetosomes and SPION@Citrate

In this example, the in vivo heating efficiency and antitumoral activityof chains of magnetosomes, individual magnetosomes, SPION@Citrate andwhole magnetotactic bacteria are compared.

Materials and Methods:

All animal experiments have been conducted after approval of a protocolexamined by the committee of the “Centre Léon Bérard, Ecole normalesupérieure, Plateau de Biologie Expérimentale de la Souris, Lyon,France”.

In vivo heating experiments were carried out on 30 nude mice at 6 weeksof age, which were bought in Charles Rivers Laboratories, Arbresle,France. To prepare tumor-bearing animals, the mice were firstgamma-irradiated. Approximately two millions MDA MB 231 human breastcancer cells in 100 μl of phosphate buffer saline (PBS) were theninjected subcutaneously both on the left and right flanks of the miceusing a syringe (26 G needle). The tumor sizes were measured usingcalipers every 3 days. The estimates of the volumes of the tumors werethen carried out using the formula V=A×B²/2, where A is the longer and Bis the shorter lateral diameter of the tumor (Sun et al., Cancer Lett.,2007, 258, 109-117). The tumors grew during a period of 21 days untilthey reached a volume of approximately 100 mm³.

Before starting the treatment, the mice were anesthetized withketamin/xylazin (100/6 mg kg⁻¹, i.p.), which resulted in a decrease oftheir corporal temperature from 37° C. down to 30-36° C. depending onthe mouse. Three mice died during the first steps of the treatment mostprobably due to an overestimation of the dose of anesthetic. Afternecropsy, the organs of these mice showed no obvious systemic congestionor infarction. Under anesthesia, the needle of the syringe containingeither chemically synthesized nanoparticles or the various types ofbacterial magnetosomes dispersed in sterile water was insertedlongitudinally into the tumors of the mice. The mice were then placedinside a coil of 6.7 cm in diameter where an alternative magnetic fieldwas applied to them. To produce the alternative magnetic field, analternative current was generated within the coil using a 10 kW EasyHeatpower supply from Ambrell, Soultz, France. The schematic diagram of FIG.10( a) shows the experimental set-up used to carry out the experiments.The measurements of the temperature were carried out using animplantable thermocouple microprobe (IT-18, Physitemp, Clifton, USA) toobtain the rectal temperature or a local estimate of the temperaturewithin the central part of the tumor. The variation of the rectaltemperature was monitored to verify that the increase of the tumortemperature was local and did not take place within the whole body ofthe mice. An infrared camera (Moblr2, Optophase, Lyon, France) was usedto obtain a more global picture of the variation of temperature of thetumor and of the tumor environment. The cross section through which thetemperature was measured is indicated in the schematic diagram of FIG.10( b) by a line. The variations of the tumor sizes during the 30 daysfollowing the first treatment were measured both for the unheated andheated tumors.

Antitumoral activity was studied by following the size evolution oftumors grown subcutaneously on both flanks of each mouse. Mice wererandomly selected and divided into five groups. The first four groupswere treated as follows. One hundred microliters of suspensionscontaining individual magnetosomes (suspension 1 in mice 1 to 3), chainsof magnetosomes (suspension 2 in mice 5 to 8), SPION (suspension 3 inmice 10 to 13) and whole magnetotactic bacteria (suspension 4 in mice 15and 16) were administered into the tumors localized on the right flankof the mice. After injection of the different suspensions, mice weresubjected to an alternative magnetic field of frequency 183 KHz andmagnetic field strength of ˜43 mT (mice 1, 2, 3, 5, 6, 7, 8, 10, 11, 12,13) or ˜80 mT (mice 15 and 16) during 20 min. The treatment was repeated3 times at 3 days interval. For the mice, which received the suspensionof chains of magnetosomes, the magnetic field had to be reduced by ˜5 mTto avoid that the temperature within the tumor exceeds 50° C. For themice, which received the whole bacteria, the magnetic field strength hadto be increased to ˜80 mT to observe a temperature increase within thetumor. The fifth group was considered as a control group and was notsubjected to the application of an alternative magnetic field. Thisgroup was composed of mice, which received into the tumors localized ontheir right flank, 100 μl of physiological water (mice 17 and 18), 100μl of suspension 1 (mice 4, 19 and 20), 100 μl of suspension 2 (mice 9,21 and 22), 100 μl of suspension 3 (mice 14, 23 and 24) and 100 μl ofsuspension 4 (mice 25, 26, 27). Finally, the 27 tumors localized on theleft flank of each mouse were used as internal control and only receivedphysiological water.

The concentrations of the different suspensions (10 mg ml⁻¹ forsuspensions 1 and 3 and 20 mg ml⁻¹ for suspension 2) were chosen in sucha way that they yielded similar heating properties in water. Theseconcentrations represent the amount of maghemite contained in onemilliliter of water. They were estimated in three different ways, eitherby measuring the absorbance of the different suspensions at 480 nm, byweighing the amount of nanoparticles or magnetosomes afterlyophilization or by measuring the saturating magnetization of 20 μl ofeach suspension deposited on top of a substrate using SQUID magnetometermeasurement (Alphandéry et al., J. Phys. Chem. C, 2008, 112,12304-12309). These three different types of measurements yielded thesame estimate of the concentration for the suspensions containingindividual magnetosomes and SPION. For the suspension containing thechains of magnetosomes, the presence of biological material surroundingthe bacterial magnetosomes led to an overestimate of the maghemiteconcentration by absorbance and lyophilization. Therefore theconcentration of this suspension was determined using SQUIDmeasurements. For the treatment with the whole magnetotactic bacteria,the bacterial concentration injected was 10⁸ cells in 100 μl. Theconcentration of bacterial cells was chosen so that it yielded the sameiron oxide concentration than that of suspension 2.

Histological examinations were carried out in subcutaneous tumor, liver,kidneys and lungs collected 30 days after the first injection. Sampleswere fixed in 10% formalin solution, embedded in paraffin and sectionedinto slices of thickness 4 μM. The sections were stained withhematoxylin-eosin (HE) and with Berlin blue to detect the presence ofthe bacterial magnetosomes dyed in blue. Necrosis of neoplastic cells,the number of mitoses per 3 randomly selected fields at a magnificationof ×400 in non necrotic area and the amount of pigmented cells wereevaluated in pathological sections of the tumors localized on the rightflank of the mice.

In order to shed light on histological examinations and to studyinternalization of the magnetosomes within tumor cells, 5.10⁵ breastcarcinoma cells (MDA-MB-231 lines) have been seeded on microscopy slidecover. They grew during 48 hours at 37° C. in 5% CO₂. Cells were furthertreated in the presence of various suspensions of magnetosomes during 1to 24 hours in the absence or in the presence of a magnetic field of 0.6mT. Two milliliters of the two suspensions of magnetosomes, containingeither individual magnetosomes or chains of magnetosomes mixed in thecell growth medium, were used. In order to avoid too high cytoxicity ofthe cells, the iron oxide concentration of the two suspensions ofmagnetosomes was kept low at ˜130 μg·ml⁻¹. After treatment, the cellswere washed with PBS to remove the bacterial magnetosomes surroundingthe cells. The cells were then fixed using 5% of paraformaldehyde andwere incubated in the presence of a solution, which becomes colored inPrussian blue in the presence of iron. This solution contains 5%potassium ferrocyanate and 10% hydrochloride acid (equivolume). Thecells were then observed using an air objective (×100). The focalizationof the objective was adjusted to detect the presence of iron within thecells and not at the cell surface.

Results and Discussion:

In the first set of mice, the suspension containing the individualmagnetosomes was injected and the alternative magnetic field wasapplied. As a result, the temperature within the tumor mice increased by4° C. from 31° C. to 35° C. (FIG. 10( c)). After 10 min of heating,infra-red measurements showed that the temperature spread within thetumor is ˜0.5 cm, where this distance is estimated by measuring the fullwidth half maximum of the temperature distribution shown in FIG. 10( d).The evolutions of the sizes of the treated tumors are shown in FIG. 10(e). These sizes increased in mice 1 to 3 during the 30 days followingthe treatment, indicating the absence of antitumoral activity. Theincrease of the treated tumor size can also be observed in mouse 3 byexamining the set of three photographs taken during the day of thetreatment (D0), 14 days after the treatment (D14) and 30 days after thetreatment (D30) (FIG. 11( a)). The absence of antitumoral activity inthese mice was further confirmed by the behaviors of the tumors, whichdid not receive the individual magnetosomes (FIG. 10( f)). The sizes ofthese tumors increased at a similar rate than those of the treatedtumors (FIGS. 10( e) and 10(f)). When suspension 1 was injected withoutapplication of a magnetic field (in mouse 4 and in two other mice), thetumor size also increased (FIG. 10( f)). Together these results indicatethat neither the presence of the individual magnetosomes nor the heatthat they generate in the presence of a magnetic field produceantitumoral activity.

Pathological examinations of the tumor localized on the right flank ofmouse 2 further confirmed this conclusion. They showed an important massof necrotic cell in tumors collected 30 days after the first treatment.Mitoses were numerous and indicated an important tumor proliferativeactivity with an average of 12 mitoses per selected field of 300 μm² insize. The Berlin blue staining of a pathological section obtained fromthe right tumor showed the presence of diffused dark spots (FIG. 11(b)). These spots are presumed to arise from magnetosomes aggregates asshown in enlarged views (FIGS. 11( c) and 11(d)). Histological analysisof organs showed that no individual magnetosomes were found in liver,kidneys and lungs. The absence of the individual magnetosomes withinthese organs suggests that they remain localized within the tumors 30days after injection, probably because of their tendency to aggregate.

In order to study if the individual magnetosomes penetrate withincarcinoma cells, the latter were incubated in the presence of asuspension of individual magnetosomes. After 1 hour of incubation, thereare only few traces of individual magnetosomes located inside the cellsboth in the absence and in the presence of a magnetic field. After 24hours of incubation of the cells, no more traces of individualmagnetosomes were observed both in the absence and in the presence of amagnetic field. This suggests that the individual magnetosomes don'teasily penetrate within the tumor cells. When they do penetrate, theydon't remain localized within these cells for a long period of time.

In the second set of mice, the suspension containing the chains ofmagnetosomes was injected. Unexpectedly, the application of the magneticfield produced a larger increase in temperature than that observed inthe first set of mice. In 20 min, the temperature within the tumorincreased by 10° C. from 33° C. to 43° C. (FIG. 12( a)). In addition,infra-red images show a larger spread of temperatures through the tumorcross-section. FIG. 12( b) shows that the full width half maximum of thetemperature distribution is 0.75 cm, suggesting a more homogenoustemperature distribution within the tumors for the chains ofmagnetosomes than for the individual magnetosomes. By contrast to thebehaviors observed with the individual magnetosomes, the antitumoralactivity was clear in this case. FIG. 12( c) shows that the sizes of thetreated tumors did not strongly increase as it is observed for theuntreated tumors (FIG. 12( d)). The treated tumor disappeared completelyin mouse 5 and was very significantly reduced in size in mouse 6 (FIG.12( c)). The disappearance of the treated tumor in mouse 5 can be seenby examining the set of three photographs taken during the day of thetreatment (D0), 14 days after the treatment (D14) and 30 days after thetreatment (D30) (FIG. 13( a)). In addition, histological examinationsshowed that there was no remain of tumor tissues in mouse 5.Pathological examinations of the treated tumors showed that the numberof observed mitosis was low (4 in average by selected field of 300 μm²)indicating a decrease in the activity of tumor proliferation. In mouse 9and in two other mice, where the suspension of chains of magnetosomeswas injected without application of the magnetic field, the tumor sizeincreased strongly during the 30 days following the first treatment(FIG. 12( d)). This indicates that the antitumoral activity was due tothe heat released by the chains of magnetosomes when they were exposedto an alternative magnetic field. A micrograph of a tumoral tissue showsa more homogenous distribution of the chains of magnetosomes comparedwith that of the individual magnetosomes (FIGS. 11( d) and 13(b)). Inaddition, the enlargements of FIG. 13( b), which are shown in FIGS. 13(c) and 13(d), show a black region surrounding the cell nucleus. Thissuggests that the chains of magnetosomes have penetrated within thecells (a result, which agrees with the conclusion drawn in example 4).The histological examinations of the organs also suggest the presence ofsporadic chains of magnetosomes, which were detected in hepatocytes andperivascular liver cells but not in kidney and lungs. Despite theaccumulation of chains of magnetosomes in liver cells, no lesions wereobserved in liver.

In order to confirm the results obtained from the histologicalexaminations, the chains of magnetosomes were incubated in vitro in thepresence of carcinoma cells. After 1 hour of incubation, the presence ofthe chains of magnetosomes within the cells was observed more clearlythan that of the individual magnetosomes both in the absence and in thepresence of a magnetic field. For an incubation time of the cells of 24hours, the presence of the chains of magnetosomes within the cellsbecomes even more pronounced. In the presence of a magnetic field, thechains of magnetosomes are localized around the cell nucleus, whereas inthe absence of a magnetic field, the chains of magnetosomes aredispersed more randomly within the different cellular compartments.These results suggest that it may be possible to target the tumor cellswith a magnetic field using chains of magnetosomes.

In the third set of mice, a suspension of SPION has been injected in thetumors localized on the right flank of the mice. The application of themagnetic field produced a slightly lower increase in temperature thanthat observed with the chains of magnetosomes. In 20 minutes, thetemperature within the tumor increased by 6° C. from 36° C. up to 42° C.(FIG. 18( a)). The full width half maximum of the temperaturedistribution estimated by infrared measurements (0.75 cm, FIG. 18( b))was the same as that observed with the chains of magnetosomes. In thiscase, the sizes of the treated tumors decreased very strongly in mice 10and 12 (FIG. 18( c)), but histological examinations showed the presenceof peritumoral lymph nodes, suggesting that the antitumoral activity wasonly partial in these mice. In mice 11 and 13, the sizes of theuntreated and treated tumors increased at a similar rate showing noobvious antitumoral activity (FIGS. 18( c) and 18(d)). In mouse 14 andin two other mice, where the SPION were injected without application ofa magnetic field, a strong increase of the tumor size was observedduring the 30 days period following the injection (FIG. 18( d)). As inthe second set of mice in which the suspension of chains of magnetosomeshas been injected, the average number of mitosis was low (5 in averageby selected field of 300 μm²) and the necrotic activity was comparableto that observed in the control group. According to the Berlin bluestaining, the SPION, which were dyed in blue were found in liver,Küpffer cells, in macrophages and in pulmonary lymph node sinus. Thepresence of SPION in the lungs has already been observed (Zhou et al.,Biomaterials, 2006, 27, 2001-2008). It is a sign of potential toxicityand is therefore a disadvantage for the development of a thermotherapysuch as that described in this disclosure.

In the fourth set of mice, 10⁸ cells contained in 100 μl of PBS havebeen injected in the tumors localized on the right flank of the mice anda magnetic field of ˜80 mT has been applied. In these conditions, thetemperature increased by only 4° C. from 33° C. to 37° C. in 20 min. Theincrease in temperature was also observed using infra-red measurements.As in the group treated with individual magnetosomes, the sizes of thetreated tumors increased during the 30 days following the treatment.Histological examination revealed a pigmented area in the treated tumorwith a high mitotic activity (15 mitosis in average by selected field of300 μm²), indicating the absence of antitumoral activity. Nomagnetotactic bacteria were found in liver, kidneys and lungs.

From these results, we can draw the following conclusions:

-   (i) No antitumoral activity has been observed when the suspensions    containing the individual magnetosomes, the chains of magnetosomes    and the SPION@Citrate were injected in the tumors of the mice    without application of a magnetic field.-   (ii) When the individual magnetosomes were administered within the    tumors to start the treatment, a low in vivo heating capability and    no antitumoral activity were observed. This is unexpected in view of    the heating capacity observed in solution (example 2).-   (iii) By contrast, when the chains of magnetosomes were administered    within the tumors to start the treatment, a significant antitumoral    activity was observed when they were heated. This behavior may be    explained by their high in vivo heating efficiency, by their    homogenous distribution within the tumor of the mice and also by    their faculty to penetrate within the tumor cells.-   (iv) The SPION, which are currently used for hyperthermia treatment    also showed antitumoral activity. However, their antitumoral    activity was less pronounced that that obtained with the chains of    magnetosomes. In addition, the experimental data were obtained for a    suspension of SPION with an iron oxide concentration, which was    twice that used with the suspension containing the chains of    magnetosomes. For two suspensions with a similar iron oxide    concentration, one observes much lower heating and antitumoral    efficiencies for an administration of the suspension containing    SPION than for that containing the chains of magnetosomes (example    6).

Example 6

Heating Efficiency and Anti-Tumoral Activity of Chains of MagnetosomesPrepared by Cultivating the Magnetotactic Bacteria Either in the Absenceor in the Presence of EDTA Compared with that of SPION@Peg andSPION@Citrate

In this example, the heating efficiency and antitumoral activity ofchains of magnetosomes extracted from magnetotactic bacteria, which havebeen prepared by cultivating the bacteria either in the absence of achelating agent or in the presence of 0.4 μM EDTA are compared.Moreover, the heating efficiency and antitumoral activity of these twotypes of bacterial magnetosomes are also compared with those ofSPION@PEG and SPION@Citrate used by other groups to carry out magnetichyperthermia.

Materials and Method:

The experimental protocol is very similar to that described in example5, except that in this case the different types of nanoparticles wereinjected only once at the beginning of the treatment. 100 μl of the fourdifferent suspensions containing 10 mg/ml in iron oxide of the differenttypes of nanoparticles were first injected within the tumors located onthe right flank of the mice. The tumors located on the left flank of themice were used as internal control. The treatment induced by heat wasstarted by applying an alternative magnetic field of frequency 183 kHzand field amplitude of 43 mT. In one case, i.e. for the magnetosomesprepared in the presence of EDTA, the strength of the magnetic field wasdecreased below 43 mT to avoid that the temperature exceeds 50° C. Thetreatment was repeated 3 times at 3 days interval. The size of the tumorwas measured during the 30 days following the treatment to evaluate theefficiency of the therapy.

The suspensions containing the extracted chains of magnetosomes,SPION@PEG and SPION@Citrate were prepared as described in example 1.AMB-1 magnetotactic bacteria were cultivated either in the presence orin the absence of 0.4 μM EDTA and the chains of magnetosomes wereextracted following the same protocol as that described in example 1.The chains of magnetosomes prepared by cultivating the magnetotacticbacteria in the absence of EDTA are designated as “standard chains ofmagnetosomes” or CM while those prepared by cultivating themagnetotactic bacteria in the presence of 0.4 μM EDTA are designated asmagnetosomes-EDTA or CM (EDTA 0.4 μM). The magnetosomes-EDTA arecharacterized by larger magnetosomes, by longer chains of magnetosomesand by a higher heating capacity (when they are mixed in water) than theCM as shown in example 3.

Results and Discussion:

When 1 mg of a suspension containing the CM is injected within the tumorand the alternative magnetic field is applied, FIG. 14( a) shows thatthe temperature within the tumor reaches 50° C. after 4 minutes oftreatment. During the 30 days following the treatment, FIG. 14( b) showsthat the normalized tumor volume, which is averaged over the differentmice treated, increases much less than that of the volume of theuntreated tumor. For the mouse, which has been treated the mostefficiently, the tumor disappears completely as indicated by thevariation of the tumor volume in this mouse (FIG. 15( b)) and by thephotograph of this tumor taken 30 days after the treatment (FIG. 15(a)). Clear anti-tumoral activity is observed with the CM, henceconfirming the results presented in example 5. When the suspension ofmagnetosomes-EDTA is administered within the tumor and the magneticfield is applied, the temperature within the tumor increases morerapidly than after administration of the CM as observed by comparingFIGS. 14( a) and 14(c). This behavior agrees with the fact that themagnetosomes-EDTA possess a higher heating capacity than the CM whenthey are mixed in solution (example 3). However, despite the fact thatthe magnetosomes-EDTA show a better in vivo heating capacity than theCM, their anti-tumoral activity is lower. Indeed, FIG. 14( d) shows thatthe volume of the tumor treated with the magnetosomes-EDTA increasesmore than that of the tumor treated with the CM (FIG. 14( b)).

FIG. 15 depicts the behaviors of the mice for which the treatments werethe most efficient in each group. A complete disappearance of the tumorwas observed with the CM and with the magnetosomes-EDTA (FIGS. 15( c)and 15(d)), suggesting an anti-tumoral activity for the chains ofmagnetosomes of various lengths.

When 1 mg of a suspension containing the SPION@Citrate is administeredwithin the tumor and the alternative magnetic field of 43 mT is applied,FIG. 14( e) shows that the temperature increases by 4° C. in 2 minutes,which is much less than the increase in temperature observed for the CM(12° C. in 2 minutes) or the magnetosomes-EDTA (20° C. in 2 minutes). Inthis case, the volume of the treated tumor increases at the same rate asthat of the untreated tumor during the days following the treatment(FIG. 14( f)) and none of the mice shows a complete disappearance of thetumor during the 30 days following the treatment. For a typical mousetreated with the SPION@Citrate, the tumor is still there 30 days afterthe treatment (FIGS. 15( e) and 15(f)). This indicates a less efficienttreatment than that involving the chains of magnetosomes. For injectionof the SPION@PEG, the temperature within the tumor of the mice does notincrease at all after application of the magnetic field as shown in FIG.14( g) and none of the tumors decrease in sizes during the daysfollowing the treatment (FIGS. 14( h), 15(g) and 15(h)).

From these results, we can draw the following conclusions:

-   (i) When various suspensions of nanoparticles containing the same    quantity of iron oxide are administered, the suspensions containing    the extracted chains of magnetosomes show a better heating    efficiency and anti-tumoral activity than those containing the    SPION@PEG and SPION@Citrate.-   (ii) The higher anti-tumoral activity produced by the CM compared    with the magnetosomes-EDTA may be explained by a better    intra-cellular uptake of the CM than magnetosomes-EDTA. This is most    probably due to the difference in chain lengths between these two    types of magnetosomes. Since intra-cellular hyperthermia is thought    to be a more efficient mechanism of cellular destruction than    extracellular hyperthermia, this difference in internalization    between these two types of magnetosomes could explain the difference    in anti-tumoral activity.

Example 7 Biodistribution of Various Bacterial Magnetosomes in Mice

In this example, the biodistribution of various types of particles(chains of magnetosomes, individual magnetosomes, SPION@citrate andSPION@PEG) contained within the different organs of mice just after theinjection, 3 days, 6 days or 14 days after the injection is studied. Forthis study, various suspensions containing 1 mg of each type of thenanoparticles mentioned above have been injected intratumoraly, i.e.directly within the tumors of the mice.

We only show the percentage of particles within the tumors and feces ofthe mice since the particles were essentially found there. For theestimates of the percentage of particles within the tumors, two types ofmagnetic measurements were carried out (MIAtek and SQUID). In additionof these two types of measurements, the specific absorption rate (SAR)of the various particles was measured ex-vivo for the tumors heatedunder the application of the alternative magnetic field. Since the SARis inversely proportional to the amount of particles heated (see example2), this measurement enables an estimate of the quantity of particlesinjected within the tumors.

Materials and Method:

Induction of human breast tumor was carried out as previously reportedin the example 5. Briefly, 54 female Swiss nude mice of 6 week of age(Charles River, Arbresle, France) received by subcutaneous injection twomillions of MDAMB231 human breast cancer cells (Cailleau et al., J.Natl. Cancer Inst., 1974, 53, 661-674) both on the left and rightflanks. The injection of the various types of particles has been carriedout 14 days after tumor implantation. A suspension of chains ofmagnetosomes, individual magnetosomes, SPION@citrate and SPION@PEG(Micromod, Rostock-Warnemuende, Germany) has been prepared at theconcentration of 10 mg Fe/mL. 100 μl of these suspensions have beeninjected directly within the tumors localized on the right flank at thedose of 1 mg of maghemite. The amount of maghemite contained within thedifferent organs of the mice has been measured during the day of theinjection (day 0, D0), three days after the injection (day 3, D3), sixdays after the injection (day 6, D6) or 14 days after the injection (day14, D14). At the different days (D0, D3, D6 or D14), the animals wereeuthanized by cervical dislocation and the tissues or organs of interest(blood, liver, spleen, lungs, kidneys, tumor, feces) were collectedimmediately, weighted and frozen at 4° C. until analysis. First, theheating efficiency of the different tumors containing the various typesof particles and collected at different days was tested ex-vivo. Forthat, the tumoral tissue was inserted within a tube, which was thenpositioned inside a coil where the alternative magnetic field offrequency 183 kHz and field strength of 43 mT was applied during 20minutes (EasyHeat 10 kW, Ambrell, Soultz, France). The temperaturewithin the tumor was measured using an implantable thermocouplemicroprobe (IT-18, Physitemp, Clifton, USA). Second, the quantity ofmaghemite was determined using an instrument, the MIAtek®, which hasbeen developed by the Company Magnisense (Nikitin et al., 2007, J. Magn.Mater. 311, 445). This technology enables sensitive detection andprecise quantification of magnetic nanoparticles in a biological target.For the measurements with the MIAtek®, the tissues were prepared bymechanical homogenization in ultrapure water (16% of feces wet weight,i.e 16 g of feces diluted in 100 ml of PBS, 25% of tumor wet weight, 50%of kidney, lung, spleen wet weight and 100% liver wet weight). 100 μL oftissues prepared in this way were placed into the detection system(MIAtek®). The calibration was carried out by measuring the MIAtek®signal of suspensions containing chains of magnetosomes, individualmagnetosomes, SPION@Citrate and SPION@PEG mixed in water as a functionof the maghemite concentration of these suspensions, which was variedbetween 15 μg/mL and 125 μg/mL. In order to verify the estimates of themaghemite concentrations with the MiAtek®, SQUID measurements have beencarried on the samples containing the highest percentage of maghemite(the tumors and the feces). For that the saturating magnetization of thedifferent tumors and feces containing the various types of particles wasestimated. From this estimate, we could deduce the quantity of maghemitepresent in the different samples using the saturating magnetization ofbulk maghemite (80 emu/g). The estimates deduced from the MIAtek®measurements have been compared with those deduced from the SQUIDmeasurements. Finally, the different tumors containing the various typesof particles have been heated ex vivo under the application of analternative magnetic field of frequency 183 kHz and field strength of 43mT. From the heating curves, we could deduce the SAR by measuring theslopes at 25° C. and hence the quantity of maghemite contained withinthe different tumors (example 2).

The estimates of the quantity of maghemite contained within thedifferent tumors have been obtained by collecting one fifth of the totaltumor volume after homogenization of the particles within the tumors.Most probably because of a non uniform homogenization, the collectedtumor does not contain one fifth of the amount of the various types ofparticles injected. This results in large error bars in the measurementsand in some cases in the detection of more particles within the tumorthan the amount, which has been injected. However, despite of theseuncertainties, the main conclusions drawn in this study remain valid.

Results and Discussion:

FIG. 16( a) shows the biodistribution of the chains of magnetosomeswithin the tumors (estimated as the percentage of injected dose per gramof tissue) just after the injection (D0), 3 days after the injection(D3), 6 days after the injection (D6) and 14 days after the injection(D14). The three types of measurements (MIAtek®, SAR and SQUID) showessentially the same trend: the rapid decrease of the percentage ofchains of magnetosomes contained within the tumors during the daysfollowing the injection (FIG. 16( a)). Indeed more than 90% of thechains of magnetosomes have been eliminated 14 days after the injection.The chains of magnetosome were found essentially in the feces with10-15% in the feces at the first day post-injection and 15 to 20% at 3days, 6 days and 14 days post-injection (FIG. 16( b)). With the chainsof magnetosomes, the route of elimination has been shown to beessentially fecal. Only few traces of chains of magnetosomes (<0.1% ID/gof tissue) were found in the lung, kidney, liver and spleen the thirdand sixth days after the injection. No chains of magnetosome were foundin the blood. These results suggest that the chains of magnetosome wererapidly excreted in their native form.

For the injection of the individual magnetosomes, the percentage ofinjected dose (I.D.) per gram of tissue is shown in FIG. 16( c) at thedifferent days post-injection. As for the chains of magnetosomes, thereis relatively good agreement between the three different types ofmeasurements (MIAtek®, SAR and SQUID). The percentage of individualmagnetosomes contained within the tumor seems to decrease much lesssignificantly during the days following the treatment than that of thechains of magnetosomes (FIGS. 16( a) and 16(c)). As for the chains ofmagnetosomes, individual magnetosome were found in the feces but with alower percentage (between 5 and 10% at D3, D6 and D14). The route ofelimination of the individual magnetosome also appears to be (at leastpartly) the fecal excretion.

The biodistribution of chemically synthesized SPION@Citrate andSPION@PEG has also been studied. As shown in FIGS. 16( e) and 16(g), theSPION@Citrate and SPION@PEG appear to remain within the tumors 14 daysfollowing the injection. The percentage of nanoparticles within thetumors does not decrease during the days following the treatment assignificantly as with the chains of magnetosomes Moreover, SPION@Citrateand SPION@PEG have not been detected in the feces of the mice. Theseresults may be explained by the fact that the SPION@citrate and theSPION@PEG are metabolized in free iron and are therefore not eliminatedin the feces as nanoparticles. This would rather be a drawback of thesechemically synthesized nanoparticles compared with the magnetosomessince free iron can cause oxidative stress (Puntarulo et al., 2005, Mol.Aspects, Med., 299-312).

From these results, we can draw the following conclusions:

The chains of magnetosomes leave rapidly the tumor and seem to beeliminated in the feces. Both of these properties are favorable for thedevelopment of the thermotherapy described in this disclosure. We couldtentatively explain this behavior by the fact that the chains ofmagnetosomes do not strongly aggregate. By contrast to the chains ofmagnetosomes, a large percentage of individual magnetosomes remainwithin the tumor 14 days post-injection, which suggest that the organismmight find it more difficult to eliminate them rapidly. We couldtentatively explain this behavior by the fact that the individualmagnetosomes aggregate. A large percentage of the chemically synthesizednanoparticles (SPION@Citrate and SPION@PEG) remain within the tumor 14days post-injection and none of them is found in the feces. Thissuggests that these chemically synthesized nanoparticles don't rapidlyleave the tumors and that they metabolize in iron and/or that they areeliminated in the urines. These features make them potentially lessattractive drug candidates than the chains of magnetosomes.

1-23. (canceled)
 24. A method for treating a cell or tissue of interestin a subject in need thereof by using a treatment induced by thegeneration of heat in which chains of magnetosomes isolated frommagnetotactic bacteria are used and where these chains are subjected toan alternative magnetic field to yield the generation of heat.
 25. Themethod according to claim 24, wherein the cell or tissue is a tumor cellor a tumor.
 26. The method according to claim 24, wherein the chains ofmagnetosomes contain at least 2 magnetosomes.
 27. The method accordingto claim 24, wherein the magnetosomes contained within the chainspossess sizes lying between 10 and 120 nm.
 28. The method according toclaim 24, wherein the chains of magnetosomes have been obtained frommagnetotactic bacteria that were cultivated in a growth mediumcontaining iron and/or another transition metal.
 29. The methodaccording to claim 24, wherein the chains of magnetosomes have beenobtained from magnetotactic bacteria that were cultivated in a growthmedium containing a chelating agent.
 30. The method according to claim29, wherein the chelating agent is chosen among the bisphosphonates,rhodamine and EDTA.
 31. The method according to claim 24, wherein thechains of magnetosomes possess an agent bound to the magnetosomes and/orincorporated within the magnetosomes, which is used to visualize thechains of magnetosomes.
 32. The method according to claim 31, whereinthe agent is a fluorophore.
 33. The method according to claim 32,wherein the agent is a fluorophore and a chelating agent.
 34. The methodaccording to claim 24, wherein the chains of magnetosomes areencapsulated within a vesicle.
 35. The method according to claim 34,wherein the vesicles are used in combination with an active principle.36. The method according to claim 24, wherein the treatment of the tumorcells or of the tumor is hyperthermia.
 37. The method according to claim36, wherein the temperature of treatment is lying between 37° C. and 45°C.
 38. The method according to claim 24, wherein the treatment of thetumor cells or of the tumor is thermoablation.
 39. The method accordingto claim 38, wherein the temperature of treatment is lying between about45° C. and about 100° C.
 40. The method according to claim 24, whereinthe frequency of the magnetic field is lying between 50 and 1000 kHz.41. The method according to claim 24, wherein the amplitude of themagnetic field is lying between 0.1 and 200 mT.
 42. The method accordingto claim 24, wherein the magnetic field is applied during a time periodvaried between 1 second and 6 hours.
 43. The method according to claim24, wherein the heating process is repeated.
 44. The method according toclaim 24, wherein targeting of the tumor(s) or tumor cell(s) by thechains of magnetosomes is carried out by using a magnetic field.
 45. Themethod according to claim 24, wherein targeting of the tumor(s) or tumorcell(s) is carried out by using an antibody and/or a folic acid and/or aPEG bound to the chains of magnetosomes or to vesicles containing them,which specifically recognizes the tumor(s) and/or tumor cell(s).
 46. Themethod according to claim 24, wherein the alternative magnetic field isapplied to improve the penetration of the chains of magnetosomes in thetumol cells.
 47. A kit comprising chains of bacterial magnetosomes and adevice, which is able to generate an alternative magnetic field.
 48. Thekit according to claim 47, wherein the chains of magnetosomes areencapsulated within a vesicle.
 49. A method for producing chains ofmagnetosomes in magnetotactic bacteria wherein the magnetotacticbacteria are cultivated in a growth medium which contains a source ofiron and a chelating agent, which method enables to increase the size ofthe magnetosomes and the length of the chains, as well as the heatingproperties thereof.